Methods in Biochemistry
Methods in Biochemistry BIOCHEM 660
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Popular in Biochemistry
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NMR STUDIES 0quot STRUCTURE AND FUNCTION OF EIOLOGICAL MAEROMOLEULES uhellaume Daemher muz w m Wm mm 1m mum 2m an m 2mm Sum ma mmkxwmu sm mam knew151m 121ml DAWN m mmonumou quotmmxmemmymxmsmmm mm m mm mm 62mm 3 an m was a mkmxm mm mw m mamm ms mxuMCMWS mnempm n m be mm 3 1 mm Comm mums my m mum 1 q am n pr byz tummy m a pm mmm 9 mm 1 pm mm mum m mm may mam am a the m asua ssnld o mamam mamkmeuzawsu mmz mmlmmeommmmms mhmyamahe Sauna9 1 am was p m mm m mama mm mm mam Wm mlunlumn m me We me me puma an mm 5mm onmzmsm sownon mm mm the send at mmmkulx um wmm m mm mm gamma mmm Namw m mum use MEI m tannn EW mm M21 my mum n m use mammal mmwm 9 m mesa aim Bummezmuamem and m me mammhgme 7amme Summmm 3pm szmw mmm mzm m 5me am by mm mm M me We mues m 3mm immunsynnunly mm m lupmmom mummmmmmel Minimum wan mg madam mwm mxappala lsu zm yduna yng nhear my mmawl h 235 many1451 me k mwWmwm 1 I L m U x 5m max mm 22s 5 VW MN WV WV WV m kwla im W M mu m mm W n MN iamau hmmmmm mm n m many mung m we cm me ohEumnsolFxgulSS s l a W and Fgue gaming am hgezmlmluum ma gm magma loammmenngfm mmmsmm 39Ilmwas gunmen mn nuad by mm at gs 51 dentin Bum mammsmm egg 9 VllEa Xue wmzakuhmnsolme WCMKws my me mmmm mm wmsmmeulmlspman m nmum mu an Mme my 1 may mm mgmmmmmm mwwmgmeay mm mm Henriksmug 1 hummus same 2 2 a K 7 s lt3 33 am a i E k F WWW 2 a Wm WM Figure 8 Local ring current eld around aromatic rings in solution induced by an ex ternal smtic magnetic eld The shape of the ring current eld is indicated by the red doublecone and by broken magnetic eld lines The minussign indicates that the NMR lines of hydrogen atoms located in side the cone in the threedimensional pro tein structure are shifted up eld to the right in the spectra of Figure 5 whereas for atoms oumide of the cone the shifts are down eld proteins and the presence of the heme iron Figure 9 was appealing in view of my background in inorganic chemistry As an additional advantage low resolution crystal structures for several hemoproteins were already available in 1967 It turned out that for several years nearly all my research projects were focused on the heme iron and its coordinatively linked ligands and on the nonbonding interactions of the heme groups with their immediate envir onment in the hemoproteins I 5A CH CHa CH3 Em 5 Hac Y CHHJ 3 H u L If hll J cm Fe agn F M H 3 1 4 CH3 Figure 9 Chemical structure of heme c which is the prosthetic group in cyto chromes c The red arrows connect roups If 2 of hydrogen atoms in the covalent stgfucture CH2 CH2 of heme c that are separated by a suf cient ly short distance to be connected by nuclear C00 C00 Overhauser effects NOE 240 mam t m away Mumps mmMgzmm mamm a 3er a helm7 am 7mm mum Mn MMMWMN mumsem m 2 337 xi amu gl Wmf n 11mwuhnmmuupdmmw mnp up cm m mmmmnnnw n mummmmmwmim mmhmmw m m mm t 2 I 39 le Htv39l k Her w thunne Unit M 2 Maul l I rc Mnums I lulu ix MH E Mnulas L I 9 1 KWJE n Jg I U1 ll 23D Mfr EH n kh N xy39ukL Figure 12 Changes with time in the 1D 1H NMR spec quot mm of Cytochrome c after reducing the fern39c cyanide complex with dithionite 220 MHZ 9 C sol vent 2H20 0 l 2 3 4 5i1pm chrome c contains a methionine side chain ofits polypeptide chains in one of the axial heme iron coordination sites Upon addition of KCN to the protein solution this methionine is replaced in the binding site on the heme iron by a cyanide ion and therefore moves out of the heme ring current eld The completion of this reaction can readily be monitored since different patterns of wellresolved resonance lines are present in the H NlVER spectra of ferricyto chrome c and its cyanide complex respectively After reduction of the heme iron to the ferrous state the cyanide complex is thermodynamically less stable than the native form of cytochrome c and in a slow reaction the cyanide in the axial coordination site of the heme iron is again replaced with the natural methionine ligand The return of the axial methionine into the heme ring current eld was monitored in real time Figure 12 by the appearance of its typical high eld lines in the 1H NlVER spectrum Figure 10 This experiment resulted in the identi cation of the nature of this axial ligand in the native ac tive form of cytochrome c and further yielded information on the relative thermodynamic smbilities of the four different structures in Figure 11 As a sideline comparison of the gures 10 and 12 illustrates the influence of various experimental factors on the appearance of the NlVER spectra The higher resonance frequency and the high temperature used in the experi ment of Figure 10 which was recorded around 1980 with Fourier transform spectroscopy resulted in narrower and more clearly separated peaks in the region 0 to 5 ppm than the experiment in Figure 12 which was recorded in 1968 with 39 V V NMR 1 at low Other early experiments based on the observation of wellresolved reso nances in the 1H NMR spectra of hemoproteins resulted in the characteriza 1 tion of conformation changes during the oxygenation of myoglobin and he 242 quotWW mm mg n m mm 5 mm a mam 3m mememmmuumu mg mam w pm my ampxanne as r2331 am not was 7 2m x m 5mm in z meolme gunman PM me mm a my m nu mug magpie mm 32 y be e nam oummgamaummgm We Noam bake m mm m 757 m m awn516nm not won We mm 1m mu g Fxgme m mnmmnm w 5 mm quota 3mm m m b nmhi zmmmns m a 65mm mg ummmxmlmlamlg m mam a me mm WWW hug m mmm mmnmezmumu mm 3 mg m m mm a 14 no Wup Mg Mm H mm no W5x m smmmzm mnnmxdmmgmem a mug memmm mmknypm ampem mil i wanna magnum my mum ninja 1 m a M 9 yep y mm an men an at me am ma mg m Figure 14 Spin diffusion 1 Transfer of magnetization between two h dro en E 391 atoms 1 and 3in thge pres r23 ence of additional hydrogen quot atoms goes through two is N competing paythways ilel 39 the direct NOE across the I dismnce r13 and two or T 1 multiplestep spin diffusion r1 via intervening hydrogen 39 atoms Corresponding considerations apply for the analysis ofNOE buildup curves recorded with truncateddriven NOE experiments 27 With proper selec tion of the duration of the mixing period one can thus measure highly se lective 1H lH NOEs between distinct pairs of hydrogen atoms or groups of chemical shiftequivalent hydrogen atoms in proteins in solution There is a second criterion that needs to be satis ed for obtaining selective NOEs with 1D NlVER experiments ie at least one of the two lines that are con nected by the NOE must be suf ciently well resolved ie separated from all other lines in the spectrum to enable selective radiofrequency preirradia tion inset in Figure 13 The previously discussed wellresolved lines in the 1H NMR spectrum of ferrocytochrome c Figure 10 thus had again an important role in enabling us to study the spin physics in the interior of this globular pro tein with 1D 1H NMR experiments as well as to obtain novel structural infor mation The gure 15 shows a series of highly selective 1D truncateddriven NOE measurements which were used to determine 1H NMR assignments for the heme group in a ctype cytochrome Figure 9 29 2 Sequenceispeci c resonance assignments Similar to the situation in heme groups Figure 9 there are closely spaced pairs of hydrogen atoms in neighbouring residues of a polypeptide chain Figure 16 These can be connected by the observation of sequential NOEs The gure 16 illustrates that NOEbased 1H NMR assignments for a polypeptide chain can conceptually be considered as a 2step process Each amino acid residue represents a spin system 4 ie it consists ofan array of hydrogen atoms including an amide proton HN an OLproton Ha and the side chain protons which can be connected by steps over three or less cova lent bonds through the observation of scalar spin spin throughbond couplings see 4 for the exceptions represented by proline and the aromat ic side chains In contrast hydrogen atoms located in sequentially neigh bouring amino acid residues are separated by at least four covalent bonds Pairs of neighbouring residues in the sequence can therefore only be con nected via NOEs manifesting short throughspace distances such as daN and dNN Suitable 39 39 of39 39 39 quot by scalar spin spin couplings and interresidue connectivities established by sequential NOEs enable progressive resonance assignments while walking 1 11H1H 1 1 245 I 5 quotLAW 39 L mm Figure 15 1H NMR assignmenm for the a J I F heme 1group of a ctype cytochrome 4L 7 7 M Hr using H N NOEs see Figure 7 Bottom trace lD 1H NMR spectrum 5 L J of 0551 from 1 I Pseudumumls dmginum M 12000 Upper traces three lD truncateddri ven NOE difference spectra obmined with selective preirradiation indicat ed by arrows on the hydrogen atoms 3 0L and 5 see Figure 9 respec 39vely IL 360 MHZ 35 C 7H20solution The NOE peaks are identi ed with num bers indicating the substituenm at 39 mched to the corresponding por 9 I5 3 0 ppm phyrin Iing carbons Figure 9 I l Lug a JJ LL along the polypeptide backbone In other words the spin systems of two neighbouring residues can be connected by the intervening Hquot HN or HN HN sequential NOE connectivities Figure 16 1D doubleresonance experi ments with selective irradiation of the wellresolved amide proton resonances between 8 and 10 ppm in folded BPTI Figure 5 thus yielded assignments for most of the residues in the regular secondary structure elements Figure 17 30 Further assignments were not possible because the other regions of the polypeptide chain were not represented by wellseparated 1H NlVER lines that could have been selectively irradiated in 1D NlVER experiments 3 Twodimensioml 2D NIWR With the introduction of 2D NlVER experiments and subsequently 3D and 4D NMR experiments NMR studies of biological macromolecules evolved from intellectually stimulating science to a practical approach for protein structure determination The foundations of multi dimensional NlVER have been pre sented in the Nobel lecture by Richard R Ernst Here I only want to Fi re 16 Se uential 1H NMR assi Vanna Alan39nei 39i mi of progeins The drawing shcgyiws the chemical structure ofa valine alanine dipeptide segment in a polypeptide chain The dotted lines connect groups of hydrogen atoms that are separated by at most three chemical bonds and can therefore be connected using scalar spin spin couplings The broken arrows link pairs of hydrogen atoms in neighbour ing amino acid residues that are sepa rated by short throughspace dis mnces dmN and and can therefore be connected by sequential NOEs 246 mm m rn39xu nmmm Whmmmmmmmxmhy quotBaum Dunnme imma39 mamm pmmusalu g meaan mmnmn SnamlmbpmamiNm ngmlmym mugmum by amuswumm m m m r m M24 kmmmnvxmu mm mum mammmlmmme mm WW mummy mum mg qumwnnn mmwummmmmw 33mm mmnmenmms 1 WW mhmmnmhva oEQpaknmmuns k a mmwmmeuozamammmmmnxmn smesumolme gamma maan mna nswmmenummm n m W mm mm Wmqu m mm gamma p m Wammmm as mgelummu mempmxns nhau mnagmm e em mm mammummunmpxu mum mmvmm unqualam39me 5mm Sludga l mslmgmm 3 mm a W11sz ahmnm mnluxmasalegama l mimgmpa mm me quotam I uhnssglsns l me msxmmlem39mepm Mimgeulmep use W Xinguen 5mm misammmepammmmmam mm many 3 mm a mums D mnluxmasmma mam where the structure is de ned with high precision by the NlVER data whereas structurally disordered polypeptide segments show a large dispersion among the members of the bundle as exempli ed by the two chain ends of the Antennapedia homeodomain in Figure 1 In the absence of longrange NOE distance constraints a properly functioning algorithm for the structure cal culation will sample the entire conformation space that is accessible with the given length of the polypeptide chain as exempli ed by the unstructured tail of the bovine prion protein in Figure 4 NlVER IN STRUCTURAL BIOLOGY Standard protocolfor NIWR structure determination of biological macromolecules The protocol for NlVER structure determination includes the preparation ofa homogeneous protein solution the recording and handling of the NlVER da ta sets and the structural interpretation of the NlVER data Table l The tech niques used in 1984 for the structure determination of bull seminal pro teinase inhibitor IIA are listed in Table l the four steps of the structural interpretation III in Table l were performed separately although the result of the rst round of constraint collection and structure calculation was sub sequently used for additional checks on the sequencespeci c resonance as signments as well as on the collection of conformational constraints Since 1984 the protocol outlined in Table 1 has been used in over 3 000 NMR structure determinations of proteins and nucleic acids and greatly improved experimental techniques have been incorporated into this general scheme Major advances in the experimental techniques for NMR structure de termination were spurned by the introduction of methods for the produc tion of recombinant proteins labelled with stable isotopes in particular Table Ii Standard protocol for NMR structure determination of proteins Step 1 BUSI IIA b I Sample preparation Protein isolated from natural source natural isotope distribution 16 mM solutions in H20 and in ZHZO respectively II NMR spectroscopy 2D 1H NMR IIIa Resonance assignments Sequential NOEs IIIb Conformational constraints 1H1HNOEs UHNH ZLB IIIc Structure calculation Metric matrix distance geometry IIId Structure re nement Restrained energy minimization a The structuml interpretation of the NMR data 111 is somewhat arbitran39ly divided up into four steps in practice one goes though multiple cycles of collection of conformational constraints 1111 and structure calculation IHc and the completion ofthe sequencerspeci c assignments Illa as well as the structure re nement Illd may also be part of this iterative approach This column lists the techniques used in the rst structure determination of a globular protein in 1984 43 b 253 mum whmm m um Fixm4quotva kman N may DmWMMM m m tm x m mm Mm monk mqmgmmBmm m menyle msopaai me older mm mum mm mm me am me my mums m 3mm man z ya man a 457 mmmmu muncwmmmm a nu me my we met acmean ma mm 5 ohm r W a me m m mmmmmm Mummy um P1 mmmhamm y mmmmammwwmmrw Mun W MA w A mmpan wpmm w mm mm mm Mm m Ems managingmama 19 m mmmuumm u Mammmm m m mm mm magma 1 am mum gaming mmuppamm new is PM for individual waters may be signi cantly shorter Rapid exchange of interior hydration water molecules appears to be a general property of globular pro teins and was also observed for water molecules located in protein DNA in terfaces for example in the DNA complex with the Antennapedia home odomain Figure 2 Another intriguing NlVER observation of internal protein mobility are the 180 ring flipping motions of phenylalanine and tyrosine 54 Observation of these ring flips on the millisecond to microsecond time scale Figure 29 was a genuine surprise for the following reasons in the re ned Xray crystal struc ture of BPTI reported in 1975 the aromatic rings of phenylalanine and tyro sine are among the bestde ned side chains with the smallest temperature fac tors In each ring the relative values of the temperature factors for the individual atoms increase toward the periphery so that the largest positional uncerminty is indicated for the peripheral carbon atom on the symmetry axis through the Vi C1 bond rather than for the four 8 and e ring carbon atoms which undergo extensive movements during the ring flips Theoretical studies then resolved this apparent contradiction the crystallographic tem perature factors sample multiple rotation states about the Cu C 3 bond but they do not manifest the ring flips because the populations of all nonequilib rium rotational smtes about the C3 C1 bond are vanishingly small Although the flipping motions about the Vi C1 bond have low frequencies Figure 29 they are very rapid 180 rotations connecting two indistinguishable equilib rium orientations of the ring Similar to the exchange of internal hydration waters the ring flip phenomenon is a general feature of globular proteins ma nifesting ubiquitous lowfrequency internal motions that have activation ener gies of 60 to 100 kJM39l amplitudes larger than 1 A activation volumes of about 50 A3 and involve concerted displacement of numerous groups of atoms Combined with sequencespeci c NlVER assignments these experiments pro vide high spatiotemporal resolution for the description of rate processes in proteins In Figure 29 this is illustrated by a mapping of the frequencies for ring flips and water exchange onto the NMR structure of BPTI In addition to the ring flips and the exchange of internal hydration water molecules the gure 29 includes data on the exchange of a disul de bond between the R and S chiral states In contrast to the other two phenomena this rate process connects two different molecular structures Although all the data collected in Figure 29 have been known for more than a decade and some of them for nearly three decades no widely accept ed functional interpretation of these lowfrequency motional processes has been advanced The same holds for the conformational equilibria manifested by the protection factors governing the amide proton exchange rates in folded proteins Figures 17 and 21 Quite possibly these NMR measure ments are ahead of their times and represent a source for future novel in sights into structure function correlations in proteins 257 m a mum mnmmmmup n Wm M mummygm n7 mum mmmm mmnmxumunw u m WWquot 1 in PM mm mm many a m m m WM 10me a 3 m my nm mm imam a km a 4 a m 339 mw mmmwmnmwsan g9p 9 OU39ILOOK 10 um mummus m nwmummn mummy anrzomcs m me mumw gammy m th gamma mm mm new mm mum mm 3mm m mlumn ludhan mm 1 mm 3 mm mhsup 1 abmn 10 um Fgue 2 121327 a 12sz m mm 1 mm u Mm a IW39H alvmldaz and mm a 1 a mu m m Edam mlmEomummmxm mm an mvenhemmu Wigwam m u daagmsonqnds d xgmel ngumy Algwyezsagu m m Vau enga l mememmgempwamm lumn mm mm zm d he mum a mu me at lemmlwpnmnai wmm mom m Asalumsnum me n am pm my 1R05qunel imam yuan mm m mm 94 131 M m 3 m at me mum ma a g mmmwqumemyxnmhge DWIROSYEnheqmnnvelynmmnaihyme lnmgzmmnA mmhamm mnun ok qm my me appeammolmlumn mxpanxsmmewman manve m 28 mm a ammumumw Mumumm wwwmmm maunmmnnm mm mumFquot mmuamatmwmunmm mu m hmwnmmwymMmMmmpu m m mm a mwhmmumm gay moimenwkule hemmedwmmg yymnmwnm amgamewm mm Mum nmm r wmamuwmm m m auxlam mmwwmmm m p hmwmmnm mumwwummm my w ammmmmmn wxqumnmh hm Q nm mgmuuyxmmm amumy mmw m wwwmm mm 7194 mm mm 1 mm mmnm PM mummmmnmwmm Funnmg my mama a p munm aimeme use Mm m mum m mm WW 1 m mu m memxmolmemltlupumn haES mm m mummm Fgue m memmxpm mmvehn 95 an mun mm an mm 5 WWquot Mmmmwmmem 4 ammun Mmmeapmmmxwmew Magnumme mmmmmumpmummnmmquotwku m mam n w uhmmmnumsmu m hump nu pannmrmunmm human mdmtmmmmewsmumme m minm n a m m Mmmmmm mane M xelzxamnzanhe m 1 m m mg mm H ame mg a mummm mama y mmwlmmw 25A cm 1 m m mhv maxim m mmmmm unencmmM leash sm mm mm o mm mm helm me an m m at magnumy mwmsmmam maximum m a my me ame me um Wm s a smvely WWW m m Emma m 3pm m mg m hmnhe new Emmmhemm mm Fu nhw m me me mg Puma mm in mg m be am am v Fwsms 55 367 comm m Ebb mp3me mag mm m KW gm 1 a wannapplmzhlux ms ymmolmamda mm mm mmnlw m 3mm m m a we yams mm m m use mm amwnmmmmnnuhva umn nemadsmmyezws 752 E mER winamg iua pgam ai ir z a 3 m a as a E Easia 339 a a was an ma m EVER minim ENEE 512 Euzma soi a 5535 E a a 3 E i m a 233 mm Equot a 5 i a 54 ninxa s a in p 4 9 l mg 1739 ii 5313 tn 3253 muiuawmu 353 2535 of our research projects also depended critically on nancial resources I would like to acknowledge the ETH Zurich the Swiss National Science F 39 theV 39 39 furT 039 undT 39 KTIBruker Biospin AG and the Scripps Research Institute in LaJolla CA USA for their support REFERENCES Qian YQ Billeter M Otting G Muller M Gehring and Wuthn39ch K 1989 Cell 59 573 580 The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution comparison with prokaryotic repressors 2 Otting G Qian YQ Billeter M Muller M Affolter M Gehn39ng and Wuthrich K 1990 EMBO 9 3085 3092 Protein DNA contacts in the structure of a homeodomain DNA complex determined by nuclear magnetic resonance spec troscopy in solution 3 Lopez Garcia E Zahn R Riek R and Wuthrich K 2000 Proc Natl Acad Sci USA 97 8334 8399 NMR structure of the bovine prion protein 4 Wuthrich K 1986 NMR ufPruteim and Nucleickids Wiley New York 5 Wuthrich K 1995 Acta Cryst D 51 249 270 NMR this other method for protein and nucleic acid structure determina 39on 6 Wuthrich K 1995 NMR in Stmctural Biulugy A Cullectitm ufPapers by Km Wdtlm39ch World Scienti c Singapore 7 Wuthrich K 2001 Nature Smut Biuli 8 923 925 The way to NMR structures ofpro teins 8 Saunders M and Wishnia A 1958 Arm NY Acad Sci 70 870 874 Nuclear mag netic resonance spectra of proteins 9 Jardetzky O andJardetzky CD 1958 Biol Chem 233 383 387 Proton magnetic resonance spectra of amino acids 10 KowalskyA 1962 Biuli Chem 237 1807 1819 Nuclear magnetic resonance studies of proteins 11 Mandel M 1965 Biuli Chem 240 1586 1592 Proton magnetic resonance spectra of some proteins 12 McDonald CC and Phillips WD 1967 Amer Chem Sac 89 6332 6341 Manifestations of the tertiary structures of proteins in highfrequency nuclear mag netic resonance 1 Wuthrich K 1969 Pruci Natl Acad Sci USA 63 1071 1078 High resolution proton nuclear magnetic resonance spectroscopy of cytochrome c 14 Shulrnan RG Ogawa S Wuthrich K Yamane T PeisachJ and Blurnberg WE 1969 Science 165 251 257 The absence of heme heme interactions in hemoglo bin 15 Davis DG Lindstrom TR Mock NH BaldassareJJ Charache SJones RT and Ho C 1971 Mali Biuli 60 101 111 NMR studies of hemoglobins V1 heme proton spectra of human deoxyhemoglobins and their relevance to the nature of co operative oxygenation of haemog o in 16 39 and Ho C 1968 Amer Chem Sac 90 2700 270 Kurland RJ Dav1s D G 1 Pararnagnetic proton NMR shifm of metmyoglobin methemog obin and hemin de riwtives 17 Wuthrich K 1970 Stmcture and BandingS 53 121 Structural studies of hemes and hemoproteins by nuclear magnetic resonance spectroscop 18 Shulrnan RG Glarum SH and KaIplus M 1971 Mali Biuli 57 93 115 Electronic structure of cyanide complexes ofhemes and heme proteins 19 Wuthrich K 1976 NMR in Biulugical Research Peptides and Prateins North Holland Amsterdam 264 N o N N3 09 N3 9 N3 0 N3 517 09 o 09 09 N 09 09 09 9 09 m 09 0 09 517 NoggleJH and Schirmer R E 1971 The Nuclear OverhauserE ect Academic Press New or Gibbons WA Alms H Bockman RS and Wyssbrod HR 1972 Biachemistry 11 1721 1725 Homonuclear INDOR spectroscopy as a 39 39 an analyz ing proton magnetic resonance spectra of peptides and as a basis for determining sec ondary and tertiary conformations of complex s Gupm RK and Red eld AG 1970 Science 169 1204 1206 Double resonance NMR observation of electron archange between ferri and ferrocytochrome c BalaraIn P Bothner By AA and DadokJ 1972 Amer Chem Sac 94 4015 4017 Negative nuclear Overhauser effects as probes of macromolecular structure Solomon 1 1955 Phys Rev 99 559 565 Relaxation processes in a system of two Spll lS Kalli A and Berendsen 1976 Magn Remn 24 343 366 Proton magnetic relaxation and spin dif sion in proteins Gordon SL and Wuthrich K 1978 Amer Chem Sac 100 7094 7096 Transient proton proton Overhauser effects in horse ferrocytochrome c Wagner G and Wuthrich K 1979 Magn Remn 33 675 680 Truncated driven nuclear Overhauser effect TOE a new technique for studies of selective 1H lH Overhauser effects in the presence of spin diffusion AbragaIn A 1961 Piinciples quuclearMigndism Clarendon Press Oxford Keller RM and Wuthn39ch K 1978 Biachem Biophys Res Cumm 8 1132 1139 Evolutionary change of the heme c electronic structure ferricytochrome c551 from Pseudumunas deruginum and horse heart ferricytochrome c Dubs A Wagner G and Wuthrich K 1979 Biochim Biophys Acid 577 177 194 Individual assignments of amide proton resonances in the proton NMR spectruIn of the basic pancreatic trypsin inh1bitor Ernst RR 1992 Angem Chemie 71m Ed 31 805823 Nuclear magnetic resonance Fourier transform spectroscopy Wider G Macura SAnil Kumar Ernst RR and Wuthrich K 1984 Magn Remn 56 207 234 Homonuclear twodimensional 1H NMR of proteins experimental pro cedures Ernst RR Bodenhausen G and Wokaun A 1987 Principles 0f Nuclear Magnetic Remnance in One and TM Dimensiuns Oxford University Press Oxford Wagner G and Wuthrich K 1982 Mal Biol 155 347 366 Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra basic pancreatic trypsin inhibitor Wagner 0 AnilKuInar and Wuthrich K 1981 Eur Biachem 114 375 384 Systematic application of twodimensional 1H nuclear magnetic resonance techniques for studies of proteins 2 combined use of correlated spectroscopy and nuclear erhauser spectroscopy for sequential assignments of backbone resonances and elu cidation of o ypeptide secondar structures Billeter M Braun W and Wuthrich K 1982 Mal Biol 155 321 346 Sequential resonance assignmenm in protein 1H nuclear magnetic resonance spectra computa tion of sterically allowed proton proton dismnces and smtistical analysis of proton proton distances in single crystal protein conformations Wuthrich K Billeter M and Braun W 1984 Mal Biul 180 715 740 Polypeptide secondary structure determination by nuclear magnetic resonance obserwtion of short proton proton distances Pardi A Billeter M and Wuthrich K 1984 Mal Bial 180 741 751 Calibration of the angular dependence of the amide proton C proton coupling constanm flaw in a globular protein use of law for identification of helical secondary struc means of sin 1 ture Wuthrich K Wider G Wagner G and Braun W 1982 Mal Bial 155 311 319 Sequential resonance assignments as a basis for determination of spatial protein struc tures by high resolution proton nuclear magnetic resonance 265 4 o 4 4 N3 4 m 4 0 4 o m o m m N m 09 m m m 0 m o 0 o 266 Braun W Bosch C Brown LR Go N and Wuthrich K 1981 Biuchim Biophys Acta 667 377 396 Combined use of proton proton Overhauser enhancements and a dismnce geometr a1 orithm for determination ofpolypeptide conformations ap plication to micellebound gluca on Havel TE and Wuthrich K 1984 Bull Math Bial 46 673 698 A dismnce geometr program for determining the structures of small proteins and other macromolecules from LluLlcdi 39 F39 H lH proinnities in solution Havel TE and Wuthrich K 1985 Mal Biul 182 281 294 An evaluation of the combined use of nuclear magnetic resonance and dismnce geometry for the deter mination of protein conformations in solution Williamson MP Havel TE and Wuthrich K 1985 Mal Bial 182 295 315 Solution conformation of proteinase inhibitor 11A from bull seminal plasma by 1H nu clear magnetic resonance and distance geometry Wagner G 1993 Bimrwl NMR 3 375 385 Prospects for NMR of large proteins Kay L E and Gardner KH 1997 Curr Smut Biul 7 564 570 Solution NMR spectroscopy beyond 25 kDa A and Grzesiek S 1993 Accuums Chem Res 26 131 138 Methodological advances in protein NMR Wider G 1998 ng Nucl MagiL Ream Spec 32 193 275 Technical aspecm ofNMR J 1 with hiolociral and studies ofhydration in solution Otting G and Wuthrich K 1990 Rev Biophys 23 39 96 Heteronuclear lters in twodimensional 1H1HNMR spectroscopy combined use with isotope labelling for studies of 39 and 39 39 39 Guntert P 1998 Rev Biophys 31 145 237 Structure calculation of biological macromolecules from NMR data Moseley HNB 34 Montelione GT 1999 Curr 0pm Struct Biol 9 635 642 Automated analysis of NMR assignmenm and structures for proteins Herrmann T Guntert P and Wuthrich K 2002 Bimrwl NMR 24 171 189 Protein NMR structure determination with automated NOE identi cation in the NOESY spectra using the new software ATNOS Luginbuhl P and Wuthrich K 2002 Prng Nucl MagiL Ream Spec 40 199 247 Semiclassical nuclear spin relaxation theory revisited for use with biological macro molecules Otting G Liepinsh E and WuthIich K 1991 Science 254 974 980 Protein hydra tion in aqueous solution WuthIich K and Wagner G 1975 FEBS Lat 50 265 268 NMR investigations of the dynamics of the aromatic amino acid residues in the basic pancreatic trypsin inhibitor Shuker SB Hajduk PJ Meadows RP and Fesik SW 1996 Science 274 1531 1534 Discovering highaf nity ligands for proteins SAR by NM Pellecchia M Sem DS d Wuthrich K 2002 Nature Rev Drug Disc 1 211 219 NMR in drug discovery Pervushin K Riek R Wider G and Wuthrich K 1997 Pruc Natl Acad Sci USA 94 12366 1 2371 Attenuated T2 relaxation by mutual cancellation of dipole dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures ofvery large biological macromolecules in solution Salzmann M Pervushin K Wider G Senn H and Wuthrich K 1998 Pruc Natl Acad Sci USA 95 13585 13590 TROSY in tripleresonance experimenm new per spectives for sequential NMR assignment of large proteins Fernandez C Hilty C Bonjour S Adeishvili K Pervushin K and WuthIich K 2001 FEBS Lm 504 173 178 Solution NMR studies of the integral membrane pro teins Ome and OmpA from Escherichia c0li 39 R Wider G Pervushin K and WuthIich K 1999 Pruc Natl Acad Sci USA 96 4918 4923 Pola1ization transfer by crosscorrelated relaxation in solution NMR with very large molecules Bax an 0 0 N 0 09 0 9 FiauxJ Bertelsen E Horwich A and W thrich K 2002 Nature418 207 211 analysis of a 900K GroEL GroES complex Pellecchia M Sebbel R Hermanns U W thrich K and Glockshuber R 1999 Nature Smut Biol 6 336 339 Pilus chaperone FiInC adhesin FimH 39 mapped by TROSY NMR Frickel EM Riek RJelesarov 1 HeleniusA W thrich K and Ellgaard L 2002 an Natli ALIml Sci USA 99 1954 1959 TROSY NMR reveals interaction between ERp57 and the tip of the calreticulin Pdomain Fernandez C Hilty C Wider G and W thrich K 2002 Pram Natli Acadi Sci USA 99 13533 13537 Lipid protein interactions in DHPC micelles containing the inte gral membrane protein Ome investigated by NMR spectroscopy interactions 267 Protein Characterization Via Mass Spectrometry Ed Huttlin PhD Biotechnology Center Room 2240 2628732 Biochemistry 660 elhuttlinwiscedu September 22 2008 gt I dentisz Locate protein unknown modi cations 0 use w proteins 391 n I 6 0 2quot Pnnsnnalase Find bloodborne proteins that signal tumor development Overview Introduction to biological mass spectrometry Identi cation of peptides and proteins Quanti cation of peptides and proteins Mass Spectr tocharge rati Essential Components 7 Ion source transfers electrical charge to analytes 7 Mass Analyzer separates ions based on mass per unit charge 7 Detector measures and records signal Optional Components 7 additional Mass Analyzer Mass Spectrometry OPTIONAL Vacuum ometer generates gaseous ions and measures their mass os mass analyzer and collision cell for fragmentation MatrixAssisted Laser Desorption Ionization MALDI Pulsed laser Sample dried With a matrix of one of several small aromatic organic ample Ions molecules p ate When a laser is red at Extraction grid each spot energy is Mimi 2nd Mummers Nm absorbed by the matrix and ionizes sample Proteins peptides and nucleic acids are often analyzed via MALDI 2002 Nobel Prize Koichi Tanaka Electrospray Ionization ESI i 2 i m my wwmugdimmddqssuzyzwsFdzmaymgamup I Liquid solution sprayed from tip of capillary Capillary at high voltage imparts charge Solvent evaporates droplets break apart and individual nak analyte ions enter the instrument I Used With peptides proteins and small organic molecules 2002 Nobel Prize John Fenn Mass Analyzers Quadrupole fezsemi V V l mm Samvie mum m m Time of Flight TOF I ii Ion Trap Orbitrap Fourier Transform Ion Cyclotron Resonance FTICR 39 39 39 39 separatingiun based o 39 39 vaiic Each has unique performance characteristics 7 Sensitivity 7 Resolution 7 Speed ofData Collection Speci c ion detectors paired with each mass analyzer Different instruments are suited for different kinds of experiments Fragmentation of Biological Molecules Collision Induced Dissociation CID 7 Ions accelerate and collide with inert gas eg N2 7 Ions absorb energy during collisions 7 Eventually ions break COLLISION CELL VOLTAGE Measuring Masses of Intact Ions OPTIONAL Precursor Ion Scan MS Survey Scan Data A Mass Spectrum in i ii r i am 5am 500 mu and gun man nan 12m 1350 um redo 15m mm tsnn mm 2m 2m Mass per Unit Charge mz VOLAETVLSOMLGDANADNGR Determining Charge State r hm K Mass difference between peaks 1 Da g compare RC and BC g mZ difference 05 mZ difference 0333 i 1z05 1z0333 Inlmrmuu 22 MS Survey Scan Data A Mass Spectrum iiih ii M an r A a man rrnu39mu um um rsoo 180D mm mm ma 2mm 2mm Mass per Unit Charge mz Determining Neutral Mass vomzrvtsmiemmmise mumquot During ronrzanon the pepnde picks up H ions one for each charge M neutral mass mZ obs masstocharge ran o 3 MtZH mZ Z M 23482202 f M2H11741 2 M234613Da uer 39 a in my im in B M2H 23481 Measuring Fragmentation Patterns OPTIONAL Vacuum 1 Select ion in rst mass analyzer 2 Fragment the species 3 Measure mass to charge ratios of fragments in second mass analyzer MSMS Scan or Product Ion Scan Intensity Intensity MSMS Fragmentation Data MS Survey Spectrum 1 1 Lilli l 39m rm nw39um39uw Man 1 v Mass per Unit Charge mz r we who may 1900 zdw39zmo Product Ion Spectrum for 8423 mz u and J r l l 1 100 1200 13m um mm 909 mm Ha Mass per Unit Charge mz HPLC and Mass Spectrometry ESlMS can be interfaced directly with an HPLC Need MSfriendly LC conditions 7 Solvents 7 Flow rates 7 Volatile buffers Online HPLC separation allows MS characterization even of very complex samp es HPLC ESI COLUMN SAMPLE Mass Spectrometer X Protein Characterization Via MS 0 Most common 7 Identi cation of peptides and proteins 7 Detection of post translational modi cations 7 Quanti cation 0 Less common 7 Structural and conformational information 0 Proteomics High throughput study of global patterns of protein expression and modi cation Strategies for Protein Characterization T09 Down Analyze intact proteins a r v BottOm U9 Digest proteins and analyze Trypsin I LEE 522 e Protein Peptides l ill r peptides Identi cation of Proteins Two primary strategies 7 Peptide Mass Fingerprinting 7 MSMS Peptide Fragmentation Genome sequence is important 7 Identification of proteins from unsequenced organisms is possible but substantially more difficult Peptide Mass Fingerprinting Mtcxmmcmvuormchcqccmcvcsvsmnnciscmmmsis rc sameRmcsmnxmvuchmcmmmcususamxu Lysozyme Pmtem mswarmcmmmqvnxmamcrwmem cmtmmmqmm Se uence WmQQLKGclsArNAGvGNvRermelcTumnranvaRAerKQIicr l Trypsin Digest PMCLVLVLLGLTALLGICQGGTGCYGSVSRIDTTGASCRT my u L VCLVRARTIAER NKYKWJKRVGEALCIEPAVIAGHSRESHACKquot KNGWGDRGNGFGLMQVDKRYHKIEG EAHIRQGTRJI mmuuQRmm wnmqoi KGGISAYNAGVGNVRSYERMDIGTLHDDYSNDWARAQYFKQHGY Each protein produces a unique set of tryptic peptides that produce a unique combination of peptide masses PeptE Klass Fingerer39 ill Predicted Mass Fingerprints 2 matches was I 5 matches I 3 matches 3 matches ilil ill ill ll l7 l Albumin I Actin Keratin m The protein is Lysozyme 1 Predict peptide mass fingerprints for all proteins in selected genome 2 Compare an observed spectrum with each predicted fingerprint 3 Identify the protein from the bestmatching predicted spectrum Peptide Mass Fingerprinting Resources Mascot wwwmatrixsciencecom Pappin DJ Hojrup P Bleasby AJ 1993 Curr Biol 36 327332 MSFit prospectorucsfedu Clauser KR Baker PR Burlingame AL 1999 Anal Chem 7114 28712882 Both programs are freely available for use online Peptide Mass No MSMS capability needed Requires pure protein Often used with SDS PAGE Cut out gel band Digest protein with trypsin in gel Extract peptides and analyze Fingerprinting Use is waning as MSMS capable instruments become more common Peptide Identi cation Via MSMS Fragmentation Imzmity yz yz b3 y3 Peptide Identi cation Via Alanine H 0 H P HZN g E oH H20 ili c c H H CH 3 893864 Da Molecular Mass 71 0371 Da Residual Mass Imzmity y yz MSNIS Fragmentation mz y3 H3quotFVDFDDPAAIEAAIDSDTRyium n inn C00quot 7Tbquot igAWATWE n77 l l l 3 l l l l i i yquot i yia quot yl 3 ya Y7 l l E l l 4 2 y yin 13900 200 300 400 500 600 mu Boo39suu 100011001 003915003913001500160617001500 mz Identifying Modified Peptides n m mo KG 5PO4 EQE SVK e 0 H l r V l I l I r m V 3E an may in Warml 39ca39wor com pH 017 111 DH 0 SH2 H2 ilirrc Nc H 5 rquot H o Unmodi ed Serine Phos hose ne Residual Mass 81032 Residual Mass 167011 Peptide Identi cation Using MSMS Search Algonthms Given the precursor mass of an 11485 03 unknown peptide and its MSMS fragmentation pattern 1 Find peptides in genome Whose mass matches the unknown 2 Calculate predicted MSMS spectrum for each matching peptide 3 Compare observed and predicted spectra 4 Return the best match and a score indicating the quality of the match Score 100 Peptide Identi cation using MSMS search algorithms SEQUEST 7 Egg McCormaek Yates 1994J Amer Sac Mass Spectrum 5 976 MASCOT FREE On a limited basis 7 www 39 sciencecom e Perkins Pappin Creasy Cottrell 1999 Electmphureszs 20 35513567 OMSSA FREE and Open Source Ge arkey Komiak Wagner Xu Maynard Yang Shi Bryant 2004 J Pruieume Res 3 958964 XlTandem FREE and Open Source 7 Craig Beavis 2004 Bzumfmmatzcs 2014661467 Spectrum Mill 7 Chalkley Baker Huang Hansen Allen Rexaeh Burlingame 2005 Mat Cell Pmmum 411941204 Hi ghthroughput Peptide and Protein Identi cation Can automate MSMS data collection and analysis 0 Typical LCMS run LTQOrbitrap r 2 hour LC gradient 7 Identify N 1500 peptides from N 600 proteins 0 Best proteomic coverage to date 7 Geobacter sulfurreducem r 3153 gene products proteins 7 91 of predicted genome 7 Ding et al 2006 Biochemica Biophysica Acta 17641198 Highthroughput Peptide and Protein Identification High Coverage for Eukaryotes 7 Arabidopsis lhaliana 7 86456 peptides from 13029 proteins 7 Surveyed six tissue types 7 Combination of 1354 LCMS Analyses 7 Baerenfaller et al 2008 Science 32093841 Peptide and Protein Quantification T A Typical Problem How do we compare blood samples of healthy and diseased mice to identify proteins Whose abundance is altered Intensity Quanti cation and Ion Intensity Ion intensity is in uenced by many factors dance 7 Innate ionization properties of each species 7 Competition With other molecules during ionization Comparing ion intensities for quantification can be misleading Without appropriate controls iill ll ill non sun am we am am loan mm mm mo um t l 5m 1600 mm tSDD mm mm mm Mass per Unit Charge mlz Isotopically Labeled Standards for Peptide Quantification n u H n vNevls7c7 rNetIC7c7r f Am lm W Y El cu mason quot0 CH Nlc an 15000t09 C 1ZC C 1SC Unlabeled Labeled 39 Similar chemical Pept39de Peptlde Properties 6011 I Little effect on ioniz 39 Easily distinguished by mass Intenx y ULL mz Stable Isotope Labeling for Proteomics In vitro Labeling A B isolate isolate protein jJ y W protein i proleolyse proteolyse derivatize derivaiize with comma Wllh heavy reagent Y reagent LCMSMS mz Metabolic Labeling B grown In heavy nutrients M t n combine grown in nutrients isolate protein proteoiyse fractionation enrichment etc LCMSMS Selected In Vitro Labeling Techniques IsotopeCoded Affinity Tags neavy reagent D87iCAT Reagent Xdeuterium lignt reagent DOICAT Reagent Xnydrogen O HN NH O 0 H O NJKI H biotin tag linker chain reactive heavy or light group Applied Biasyxtems Gygi et al 1999 Nut Biotech 17109949 Methyl Esterification OCOH I Hz NC r E l H E H 0 o Asp 6 Coo etlro CD3 x cHZ E N C39 PI H o 842 344 346 ms 550 5to1 4mz8Da 342 w 546 818 850 mz 1to1 1t05 342 M4 846 M8 850 mz Light Pep de Heavy Peptide Summary Mass Spectrometry 0 Important tool for protein characterization Identi cation Quanti cation 0 Useful for highthroughput characterization of complex protein mixtures Mass Spectrometry at UWMadison Biophysics Instrumentation Facility 7 DanylMcClaslin 7 bifmasterbiochemwiscedu Chemistry Dept Mass Spectrometry Facility 7 Maltha Vestling 7 Vestlingchemwiscedu Biotechnology Center Mass Spectrometry and Proteomics Facility 7 Amy Harms 7 harmsbiotechwiscedu Human Proteomics 7 ygephysiologywiscedu School of Pharmacy Mass Spectrometry Facility 7 Cameron Scarlett 7 cscarlettpharmacywiscedu Biochemistry 660 Methods in Membrane Biochemistry Glycnpmteln Carnal dmla Hydmfhlllc sin lulu R5 7quot lnle m Fri eln 3 anlyhnllpld Hydmplw Dir Reglon i i Hydrophobic Region Fluid Mosaic Hymn hill Memhmne in Rngon Model v Transmzmbrane Proluln Greg Kabachinski Martin Lab Kabachinskiwiscedu Processes Involving Biological Membranes SeparationSegregation Glympmleln Cgl ghi39a39i n39 v 39 Hi1 INI 39 39 39 39 Virgil V 39 mm protein speCIfICIty F Elquot ion gradients 39 Fli i ll Id 39 Wampum 39 quotSP P ExoEndocyt05is Region quotVigilquot Cell Division Maggi H m lilll Mmiygpe i Regon Signal Transduction Transmembram Protein Cell Motility and Cell Shape Fluid Mosaic Model 1972 Singer amp Nicholson Lipids serve as an impermeable barrier Proteins most otherfunction Cholesterol enhances barrier Carbohydrates glycolipids and glycoproteins Proteins vs Lipids Protein Lip d Plasma Membrane 50 50 Axon 20 80 Mitochondria 75 25 is given by weight Membrane Proteins in the Genome Hydropathy Plot E anti 5 mu siae H sapisna 2 39 Identifies hydrophobic regions l n 3 1 Kyte and Doolittle I39 O a l E Hydrophobicity of each amino acid yaxis 3 versus location in proteinXaxis 2 Predicted TM helix Hydrophobicity Alberts Molecular Biology ofthe Cell 2002 Garland Science Hydropathy Plot KyteDoolittle Hydropathy plot Arnino Transfer me 71 jggz39due f i t f m Freeenergy changes for the m 5 L153 transfer from hydrophobic to an M 4 145 e 1 mm aqueous envnronment V H 1 Leu illsl l lawEly 39 Z Hydrophilic and charged amino I acids have values at or below 0 Tim l gt ism 3 Hydrophobic amino acids have values 40 02 703 above positive values quot39139 0 7 3 0 His 4047121 Liln 4l 1 21 m as 102 Do this for all ammo aCIds In a protein fl iii 3 3 and look for hydrophobic sections of 396 1 331185 about 20 amino acids quotquotquot C Stryer Biochemistry 2001 WH Freeman KyteDoolittle Hydropathy plot 41m opium 23 e KyteDoolittle Hydropathy plot How many transmembrane domains would you predict this protein to have KyteDoolittle Hydropathy plot Is this a membrane protein Hgdr opathg Score g 41 I l 1 8 188 388 366 488 588 6218 788 Window Position w Membranes Found in the Cell You have identi ed a possible transmembrane protein does that necessarily mean it is on the plasma membrane Membranes Found in the Cell Membranes Found in the Cell Tyiges of membranes in the cell You have identi ed a possible transmembrane protein does that necessarily mean it is on the plasma membrane NO Plasma membrane makes up only a small fraction of total cell membrane ER membrane has 40 X surface area compared to the plasma membrane Other membrane bound organelles Organelle Membrane Determination Subcelluar Fractionation How to determine where your membrane can be found Look for a signal sequence Use microscopy Subcellular fractionation V 800Xg 10min Homogenate 7 H 7 15000 X g Nuclei 10 miquot 150000Xg Mito 50 miquot 1 3 ERPM 200000 x g 1 1 Ribosome gig Cuupei GM The Cell A Muleculai Annmach Subcelluar Fractionation Fleischer S Kervina M Subcellular fractionation of rat liver Methods Enzymol 197431 Pt A64 l Homogenate Mito 3 Sup Lysosomes PM Golgi microsomes Sup Peroxrsomes Nuclei Rough Smooth Subcelluar Fractionation Things to be thinking about Track your protein through the fractions by western blotting Identify the fraction with antibodies or enzyme assays PURITY OF FRACTION For example Does your mitochodrial fraction contain only proteins found in the mitochondria Membrane Protein Topology extracellular intracellular 73116 I 7j39pa II Tl39pv III Type single TM domain with Nterminus exterior side Type II single TM domain with C terminus exterior side Type III Multiple spanning TM domains Nature Vol 434 2347238 10 March 2005 Membrane Protein Topology Prerequisite in elucidating protein function is to determine its topology in the membrane Membrane Protein Topology OR Membrane Protein Topology C out OR in N Membrane Protein Topology OR Membrane Protein Topology Outside Inside 1 remove cells 2 treat with thrombin for X amount of time 3 inhibit and remove thrombin 4 detergent solublize cells and do western High MW High MW LOW MW LOW MW Membrane Protein Topology Examples of Tag gtyeusytattgn sttes restgues zymes attltatme phosphatase ataetustgease t se ta ama Detergents etergents e amphtphth umpuunds segregateg petar and apetar gematns Wdropmtc headgmup Hydrophooc Iat Detergents What are some appttcattons detergents are used m7 Detergents detergents e amphtphth eumpuunds segregateg petar and apetar gematns H at t l quot353 measurabtewatersutubttttyas munumevs ang aggregates betung to a etass gt eumpuungs known as surtaetants surfactantse muteetttes that regttee trttertaetat sttrtaee terts mmuve tun trt Hydrophoac s by absuvbmgtu trttertaees tart Detergents Detergents Detergents can fall into one of three categorizes Detergents can fall into one of three categorizes 1 Ionic cationic or anionic example SDS Which category depends on the hydrophilic extracting proteins from membranes head group of the detergent harsh and denaturing O O 5 Na Detergents Detergents Detergents can fall into one of three categorizes Detergents can fall into one of three categorizes 2 Nonionic example Triton X100 3 Zwitterionic example CHAPS 0 mild and non denaturing contain a positive and negative charge 0 commonly used in crystallography not as harsh as ionic detergents widely used in NMR structural studies OH O OCH20H2100H xSOgA HO OH Micellization CMC Detergent monomers in an aqueous solution can Detergent om ers ndergo a phase transition Selfassociation Micelle driven by the hydrophobic effect as more detergent is added Hbonding network is disrupted And water molecules must be rearranged results in unfavorable entropy detergent molecules will self associate to decrease water accessible surface wwwanatvace cum CMC Critical Micelle Concentration The concentration of detergent above which monomers will self assemble into micel es CMC is different and speci c for each detergent When working with membrane proteins a general rule of thumb is to use at least 2X the concentration of the CMC I CMC m quotminimum nuaIlra u l m l 5 m 15 5051mm mM Garavitu RM 9 a JEC mm The Micelle Detergents asymmetrical and have rough surfaces where alkyl tails are disorganized few nm in diameter have a molecular weight of less then 100 kDa dynamic structures micelles are in rapid exchange with free monomers WWW anatvace cum How does a detergent extract proteins from membranes monomers 39 micelles Detergent molecules form a torus around the hydrophobic domains lipiddetergent micelles soluble protein detergent mplex Albens MulecularEiulugv mm Cell znnz Garland Science Detergent Removal 1 Dialysis detergent solutions can be diluted below the CMC so that micelles wi disintegrate into monomers 0 good for detergents with high CMCs 2 Absorption with hydrophobic beads good for detergents with low CMCs 3 Column chromatography Com monly Used Detergents Detergent m T pe CMC mM Aggregation Brij 35 1198 N 0092 40 CHAPS 6149 Z 8 10 Deoxycholic Acid 4146 A 6 22 NP 40 603 N 50 100 155 Octyl glucoside 2924 N 18 SDS 28838 A 26 3 Triton X 100 647 N 023 75 165 Tween 20 1228 N 0059 Summary of Detergents There is NOT a set of golden rules for the use of detergents for membrane proteins Understanding of physical chemical properties is useful in deciding which detergent to use type of detergent CMC aggregation number Detergents are expensive Reconstitution To study membrane protein function in more physiological environment Higher Lipid Structure Micelle Liposome Reconstitution f 1 h gt K nump I4 Lemon K N i nun nun munn mm mam m L unum gnu quu CVTOSOL K dulurgum miccHDs monomets Reconstitution Ix a Na K Dum0 Trill quotm3 ionu munnj mm hll39 nut mum mm mom w cwosm vi amalgam lmccuns sowdim m I v mmms monomers i v quotat I s m I gt hpid dmnrgenl mmeues Reconstitution mummmlquotmaximum pmlnm 393 I quot at quotn 39 39 oquot 7 u n I V lluiddclalgeminicalies w s 39I Reconstitution mum WW pmquot mud mum mm pumrwwsun ca mw puma Reconstitution Reconstitution i H mm Va K Dump m H mmmmm mmmw mam Other Methods Electrophysiology Patch Clamping to measure current and voltage changes Capacitance measure addition or subtraction of membranes Microscopy EM freeze fracture AFM FRAP Helpful References Singer SJ Nicolson GL The fluid mosaic model of the structure of cell membranes Science 1972 175 23720731 Kyte J Doolittle RF A simple method for displaying the hydropathic character of protein J MoBio1982 1571105132 Fleischer S Kervina M Subcellular fractionation of rat liver Methods Enzymol 197431 Pt A64 l WWW anatrace com Microscopy Techniques and Applications Allyson Anding and Mark Marzinke aandingbiochemwiscedu marzinkebiochemwiscedu Clagett Dame Lab 245 Biochemistry Addition Lecture Overview 1History of Microscopy 2Microscope Basics 3Types of Microscopy 8 b Advantages Disadvantages Applications 4Campus Resources History of Microscopy Olympus vais AX 7a La 5 mm cllca was 4xrg5J camera 55 mm A am c amen Figum7 Vldea IL Camera Janssen Compound Pm OWEN Microscope circa Early 15am I Dhjeclives ias l was lei i677 i838 ideaior Leeuwenhoek Cellineory dlfjjlj ilfd compound we proposed cells microsco Protozoa l930 5 5 me erence uorescence microscopy micrOSCow developed developed 1 eleciro microscopy commercially available niip micro magnei fsu eduprlrneranatomylntroductlon niml e If r em Eyepieces Q Analyzes r Relirdalmn Comrol Pnnmmlcmgraphy Eamroller Conlroller i I e Eyepiece condenser 39 Flltevs Focus i980 s coniocal microscopy 9605 developed Basic Microscope Anatomy 7 T Tungsten Halogen Limphnue Inverted Mlnmscapa CondenserlLampnause Pillar Olympus lx7o inverted Tissue Culture Mlcroscope MillerCooled CCD Camera Eyaplece ch Prism and Phase Rlng u i r A r Prisms olilnbcuhar serve on Tube Beamsplluer JSaMllllmelar Camera System Microscope l Eleclricil Control System mm diam Mechanism Light Source Incandescent lamp Tungsten Halogen Lamphouse Ventilation Lamphouse Collector Lens t Yo Mlcmscope Pan wing Ventilation Arc lamp Arc Lamps at r ru er 7 4 J i i L If n 2quot e not Laser Common Laser System Con gurations I mo jl Cartion r 115 hire Dioxide Laser M919 in Liner r Semicondutlor Laser HeliumNeon Laser httpwwwolym pusm icrocomlprimerlanatomysourceshim Condenser Abbe Condenser Optical Pathway Microscope Abbe Slide TwoLens Condenser Condenser Aperture Diaphragm Numerical aperature NA gt close to that of the objective for the best resolution NA nsine where n imaging medium refractive index 6 12 objective angular aperature Objective Important microscope component responsible for the primary image formation 60x Plan Apochromat Objective Lateral thosepilece n aunt n Ma uiacturek Thmad FiatField Correction Nikoquot Aberration Plan Apo Correction Magnification GDX0V95 Numerical A enure Specialized 013 M Optical 101mm we 0 5 Working Properties gt 4 Distance T be Len tn Ma ni cation 6399 Cgiorcode aging Correction Range Collar Front Cover Glass Lens Element Augus g eem Assembly Figure 1 Magnification L ns Object f Image r do Magnification dido siso si image size so object size Resolution Resolution R M2nsine Where 7 wavelength n imaging medium refractive index 9 12 objective angular aperture Types of Microscopy Bright Field Phase Contrast amp Nomarski DIC Fluorescence Confocal STED TIRF PALM Electron Microscopy Bright Field Microscopy Utilizes white light to visualize an object Naked Microscopy Advantages Simplicity of setup thus lower cost No manipulation of sample Bright eld image of a live zebra sh DlsadVamaQES5 embryo at 24 hours postfertilization Stainsdyes often needed to provide contrast cells are colorless and translucent Limited in magnification and resolution mm MNWW devbiu uga EdugalleryimagessdtcycmisZLRGlpg Bright Field Microscopy Use of Stains Artificially amplifying minute differences in refractive indices w morniva rum Staining Cells Observing mRNA distribution Whole Mount In Situ Hybridization antlrsensequot sEf SE biotin hidm AAA Detect pattern of gene expression in whole embryo Tissue is xed and permeabilized n v nu r quot 39 HRP substrate of choice eg DAB Sense strand used as control for nonspeci c hybridization Staining Cells Observing mRNA distribution Whole Mount In Situ Hybridization for RAINB I expression in Rat Embryo E125 Sense Antisense ClagettDame et al PNAS 2001 Staining Cells Advantages Provides contrast to distinguish features cells are colorless and translucent Disadvantages You must fix and kill the sample Limited in magnification and resolution Phase Contrast Microscopy Amplification of minute differences in refractive indices wit 39n different regions of the cell Contrastenhancing optical technique to produce high contrast images of transparent secimens Bright Field Phase Contrast Phase Contrast Microscopy time lapse recording Differential Interference Contrast DIC aka Nomarski Differential Interference Contrast Schematic Dramatic Eyemees my improvement in resol uti o n Ability to produce m5 excellent images with relatively thick Ng g kll specimens 3 ream Lavm rmm 452m caherem Source httpwwwmicroscopyucomlarticlesdiddicindexhtml Phase Contrast vs Nomarski DIC Transparent Specimens In Phase Contrast and BIG Nomarski DIC Phase Contrast himl Phase Contrast Microscopy Advantages Increase contrast without destroying sample Specimen can remain alive Moderate cost Disadvantages Limited in magni cation and resolution Only single cell or thin layer of cells is observable Halo effect DIC Microscopy Advantages Increase contrast without destroying sample Superior resolution to phase contrast No halo effect Superior depth discrimination to phase contrast Disadvantages Limited in magni cation and resolution Only single cell or thin layer of cells is observable High cost Fluorescence Microscopy Visualization of fluorescent molecules against a dark background Applications Specifically label individual molecules proteins lipids etc within cells and tissues Fluorophores can be used directly eg green Eluorescent Erotein or conjugated to other molecules Antibodies dNTPs etc Fluorescence Microscopy httpMmminvitrogencom Fluorescent Probes Spectral Pro les of Common Fluorescent Proteins Emlsslon m o 60 n a Normalhed Speclral Pro les 5 o 0 500 450 Figure 3 Wide array of emission wavelengths Efficiency of probes based on the ability to absorb and emit photons repeatedly quantum yield Used in the detection of anatomical structures and physiological reactions in living cells a i asu 400 450 sou 550 so 550 500 650 700 Wavelength Nanamelers Wavelenglh Nanamelers Excitation and Emission Principle of Excitation and Emission Emmeu q Eluc 9391 I Banter Ultmvlolel 1W and Vlslh 2 Light Exciter Filler Fluorescent Speclmn mm WWW ulympusmicm cummumevlightandculm luuvumtvudummn html Direct uorescence myo2gfp is expressed in the pharynx Phase Contrast Phase Contrast Fluorescence Fluorescence Direct uorescence Dual labeling using GFP variant EVEquot 39 apressed in the Anchor Cell expressed in the Dislal Tip Cell Sieglveld and Kimble Staining Cells Observing DNA fragmentation Using stains to observe changes on the molecular level via secondary labeling techniqu TUNEL Assay for Apoptosis 5 HO dUTPFluorescein TdT 3 OH 180 200 bp DNA 39agment 5 H0 53 o PDUF Iuo rescein Fluorescence Microscopy TUNEL ASSAY MCF7 human breast cancer cells Fluorescence Microscopy Advantages Very sensitive Very specific timespace of expression Multiple labeling Sample can remain alive Disadvantages Photo bleaching Bleed through from other layers in tissues orwhole specimens Confocal Microscopy Visualization of fluorescent molecules in a single plane of focus to create a sharp cross sectional image Principal Light Pathways in f Pnotomuitiplier Confocal Microscopy Detector Esra lnFocus quot 0 Emission 7 Apmme Light Ray Laser Excltalion Out oiFocus Source Light Ray chhromaur Mirror I Ll hlt orrce n o e A 6mm 7 Objective Figure 1 7 Excltation Light Ray Specimen Focal PlanesE Confocal Microscopy antibody to protein A antibody to protein B merged image http medsohool umaryland eduoonfocalimagesmartinoonfooalibig ipg Confocal Microscopy Zstack Collection ofXY images along the Z axis lmages can be reconstructed for a 3D representation of sample httplwwwhihelsinki lamulAMU20Cftutlcftutjart2 3htm Confocal Microscopy Zstack Confocal Microscopy Advantages Sharper images than fluorescent microscope Less bleed through from other focal planes Can create 3D images Potential for higher resolution images when combined with other methods Disadvantages Bleaching is more ofa problem Limited to fluorescent microscopy Resolution limit is still 02 um Total Internal Reflection Fluorescence TIRF Microscopy Involves total internal reflection of fluorescence excitation light Often employed to study cellular membrane activities or the dynamics mm of actin filaments Laser 39 Excizault 9c 1 Glass n 3 5318 33 res Membra r a mu VWWV ulympus plpllklmlkruskupyZdjeclaTlRF JF39G Total Internal Relection Occurs When the Incident Angle exceeds a Critical Angle eC TRF Analysis of Vesicle Exocytosis Snell s Law nlsin 6E nzsin 62 where n refractive indices Critical Angle ecwhen 92QO C nlsin 6E nzsin 90 C nlsin 6E n2 SE sin ln2n1 ll llll mm lWpemhvsics phv39ash gsu eduhbaselphvuwlulml mml Photoactivated Localization Microscopy Photoactivated Localization Microscopy Cryusectiun at 0097 cells Exp lysusumal transmembrane prut ressing a PAVFPrtaggEd em aemg at al Science mus 313 1642745 Stimulated Emission Depletion STED Microscopy Exc Snot STED s ol Dichroic mirrors Lens Detector ase modulallon Plaza stage Hell et al Nature 440 935939 13 April 2006 Stimulated Emission Depletion STED Microscopy Neuroblastoma cells probed with an antineurofilament antibody STED image resolves neurofilament substructures f 30 nm in diameter Electron Microscopy Electron microscope uses electrons to illuminate and create an image of a specimen Two most common types of EM Scanning Electron Microscopy SEM Transmission Electron Microscopy TEM Applications of Electron Microscopy Application High resolution imaging of subcellular structures Applications of Electron Microscopy Application Specifically tag one molecule with gold particle to visualize the molecule within the cell your 7 I favorite molecule goid bead t D Rat Spinal cord With GABA distribution is Visualized by ectrori microscopy 39 tum g Electron Microscopy Advantages Resolution is in theory 010 nm only 02 pm for light microscopy Localization of specific molecules can be visualized Disadvantages Technically challenging Must coat sample with heavy metal to visualize Expensive Campus Resources Klessling dissecting microscopes ClaggetlDarne dissecting and fluorescence microscopes for tissue culture and mammals Bednarek39 fluorescence microscopes for plants Marlin confocal and TIRF microscopes UW Medical School electron microscope httplmmwmicrowiscedul Campus woe services httgwwwmicroscogywiscedu October 27th 2008 Biochemistry 660 Summer Raines smraineswiscedu Attie Lab Outline Overview of Antibodies Structure Classes Modifications Immunological Techniques Western Blotting lmmunoprecipitation lmmuno uorescence lmmunocytochemistry lmmunohistochemistry EnzymeLinked lmmunoSorbant Assay Radioimmunoassay Antibody Structure 2 light chains 1 A 2 heavy chains 1 6 e y p Bonded by disulfides noncovalent interactions Variable regions recognize antigen Fc Constant regions determine isotypefunction Technical Terminology Antigen quotInsulinquot 1 Antibody quotMouse antiinsulin 96quot 2 Antibody quotGoat antimouse IgGquot Polyclonal vs Monoclonal Antibodies Isolate Spleen cells gt Bcells Polyclona Hybridize and select hybridoma clones a l n Myeloma Cells N 1D Monoclonal Polyclonal vs Monoclonal Antibody Pros and Cons Polyclonals Resistant to epitope confirmation Inexpensive Relatively rapid Technically easy Low reproducibility CrossReactive Limited supply Monoclonals Highly reproducible Specific Limitless supply Sensitive to epitope conformation Expensive Timeconsuming Technically dif cult Antibody Modifications 1 Fluorophores fluorescein rhodamine phycoerytlirin BeadsSolid support agarose magnets ProteinA Biotin Enzymes AP HRP Western Blotting 1D method to identify a specific protein from a complex mixture BSA milk Transrerto v Visualize rwaenzo membrane gt i gt 1 Antibody 2 Antibody SDS PAGE to separate Denatured antigen Western Blotting 069 03 quot 08 035 Q AntiPKM1 1 AntiPKM2 Adapted from Christofk et al 2008 Nature 452 230234 Immunoprecipitation Isolate antigen in solution for further analysis s Antigencontaining Add 1 amlbody samp39e Add beads ProteinA SDSPAGE gt Western Blot Mass Spec Native antigen before SDSPAGE Pellet and remove supernatant Coimmunoprecipitation mycDicer Tat Tat Tat wt K51 A K41 A U E IB antimyc Dicer 5 C 5 IB antiflag Tat E 9 g lBantl ag Tat 3 1 9 2 4 Nu Ieang Immunocytochemistry Image antigen in live or fixed cells Fix Formlin PFA glutaraldehyde methanol acetone 1 Antibody 2 Antibody Cells plates on slides Red Green Merged Native Antigen Immunohistochemistry Image antigen in fixed tissue Fix Formalm PFAi giutaraidenyde methanol acetone 10 Antibody 2 Antibody Tissue sections on slides Native A ntigen Immunohistochemistry Nondiabetic Diabetic cells Insulin EnzymeLinked ImmunoSorbant Assay ELISA Qualitative or quantitative detection of antigen or antibody from a complex mixture Y Y A Y Y An ELISA Example Are You Pregnant monoclonal anllhCG Alpenzyme conjugate paiydunai antihCG Ab Ab dye substrate m dcmunslrnlu lrsl mimy sclul out ui two uuimml39s Coat we dd enzymelinked Add colored with 1n puri ed or mixture 1 Ab substrate POSITIVE OUTCOME antibody Native Antigen i dex htm706 Variations an ELISA Indirect Method Detect antibody ELISPOT Method Competitive Method K a i Detect antigen Prereact antigen1 with labeled antibody before adding to antigen2labeled well less color better binding to nt39gen1 Detect cells expressing or secreting antigen Radioimmunoassa y RIA Qualitative or quantitative detection of antigen gt Y Y Coat well 1 With antibody from a complex mixture Less radioactivity more unlabeled antigen present in e sampl Add sample puri ed Read in gamma Add saturating amounts of 125l or 5 cuunter labeled antigen Native Antigen Common Questions Asked Protein Expression Protein up or do Subcellular loca Protein binding partners Posttranslational modification ProteinMetabolic profile Disease diagnostics m E n gt Western IP wn regulation lization nannies E E El E El El El EIEIEIEI Ll quuuuq RUUIJURRE Antibodies against foreign proteins Summary of Techniques Excellent 0 Very good 0 Good 0 Fair Poor Hig Easy Cheap throughput Quantitative Western Immunoprecip ELISA Radioimmunoassay O O Q Immunofluorescence O O O itation Q Q 00 0000 Antibody Sources Commercial Molecular Probes httplprobesjnvitrogencom Jackson Labs wwwjacksonimmunocom BD Pharmigen wwwbdbiosciencescom Abcam wwwabcamcom Services V Hybridomalmonoclonal production Custommade polyclonals Outline Testing gene function Introduction Targeted gene knockouts RNA interference Biochemistry 660 Bryan Phillips September 17th 2008 btphillipswiscedu Model systems for studying gene function Mammalian cells 25000 human genes C elegans Drosophlja Arabidopsis Many of unknown function 21 st century gold rush So many genes so little time Mice Xenopus Testing gene function YFG Your Favorite Gene Testing gene function Assays Assays 7 YFG G1 Northern blot In situ hybridization M E t YFG RTpCR 4 expressed Western blot Antibody stain G2 Testing gene function Testing gene function Assays Assays Wildtype YFG mutant O I l O Wildtype Misexpress YFG 0 xx O Role in cell cycle progression 0 A X x O Reducing or eliminating gene function DNA gt ll mRNA gt ll 9 Protein gt Outline Introduction Targeted gene knockouts Target is YFG DNA Homologous Recombination Mutagenesis RNA Interference DNA Targeted gene knockout Deletion of coding region Nonsensemissense mutation Approaches Homologous recombination Chemical mutagenesis Insertional mutagenesis using transposons Homologous Recombination HR Sequencespecific recombination Exchange of DNA between strands ie Chromosomes Generation of precise genome alterations Deletions with exact breakpoints Point mutations for amino acid substitutions Gene knockout by homologous recombination g Selectable marker YFG Your Favorite Gene Selectable marker Introduce a selectable marker flanked by sequence homologous to regions flanking YF YFG replaced by selectable marker H General scheme for HR in yeast PCR amplify marker to contain YFG homology Transform yeast With 15 PCR rotiuct Homologous recombination occurs I Use selection to isolate knockout nies Q 5 colo Con rm knockout by PCR Generating knockout mice The Nobel Prize in Physiology or Medicine 2007 for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells r r Oliver Smithies Mario R Capecchi Sir Nhrtin J Evans Generating knockout mice i replace YFG with selectable marker Select for the recombination Cell culture event With Neomycln dded l j A a l Es cells Embryo cells woo woo mbryo Early mousee Inject transformed ES cells into early mouse embryo Chimeric mouse embryo Generating knockout mice I Generating knockout mice i Early mouse embryo ES cells Embryo cells I FG I FG Implant the recombinant embryos EH39W 39 quotSe e39 b39V Implant the recombinant embryos ES cells Embryo cells Coat cells Germline cells IFG IFGH DIDI Iv Cl C I I C C I I Identify YFG chimeric mice and X diimeric mouse v wildtype mouse Fc mate with widtype mice diimeric mouse CDC II BEBE II memo FG IFGor FG I X Identify YFG chimeric mice and v wildtype mouse FG mate with wild type mice A J 4 4 Identify YFG carriers and cross to m l H m each other Identify YFG homozygotes Characterizing your knockout Conditional knockouts i Characterizing knockout phenotype OXP OXP Flex re ion of interest Possible issues g 7 7 t 7 Cre recombinase mediated recombination Confirm knockout gtlt ie from tissuespecific mRNA levels promoter Protein levels Confirm specificity Neighboring genes Rescue of knockout phenotype DNA between loxF sites is excised I Conditional knockouts targeting vector Exun ioxP site homologous recombination gene ofinterest conditional knockout Creemediated recombination 2m knockout Cre expression required for knockout Tissuespeci c promoter Mouse ines may Inducible promoter already be available Gene knockouts other approaches Chemical mutagenesis eg NethyINnitrosourea ENU AT TA occasional deletions Ethyl methanesulfonate EMS GC AT occasional deletions Detection TILLING largeting induced Local Lesions IN Genomes Deletion screen using biochemical methods C elegans C elegans deletion screen Targeted gerre pecatenin gt I lt EMS Mutagenesis Targeted gene pecatenin 4 EMS WW WW w w W CLVJCLvv W gt w gt W va WW w Adult PEIEIl Larvae hennapnrudites FOR Wim genespeeiri primers Gene knockouts other approaches Transposon insertion Transposon Q 3 To Your Favorite Gene iF Gene knockouts system comparison Ordering gene knockouts Mouse International Mouse Strain Resource Organism SPGGCI Time vwvvvinformaticsjaxorgimsrindexjsp Jackson Labs E coli Fastest Days C elegans Gene Knockout Consortium wwwgrsnigacjpcelegansindexjsp YeaSt FaSter Days wwwceleganskoconsortiumomrforg C elegans Fast Wee ks Yeast Saccharomyces Genome Deletion Project vwwvsequencestanfordedugroupyeastdeletionprojectdeletions3html Arabidopsis Slower Months Drosophila melanogaster Resources listed in Nature Reviews Genetics 2005 6 179193 Mlce Slowest 1 2 years Arabidopsis thaliana TDNA insertion and knockout resources listed at httpvwvw tquot quotquotisn Outline The Nobel Prize in Physiology or Medicine 2006 Introdltmion for their discovery of RNA interference gene silencing by doublestranded RNA i39argeted gs ne knockouts RNA interference Taret is YFG mRNA Mechanism vs miRNA Harnessin lbw Andrew Z Fire Craig C Mello Potent and speci c genetic interference by doublestranded RNA in Caenorhabditis elegans Andrew FIro Sluun Xu Msvy K Montgomery Steven A Kosmaquot Samuel E Driver 64 Craig C Mello NA I L RE VOL 39l l FEBRUARY I993 Mechanism of RNAi Step 1 Mechanism of RNAi Step 2 dsRNA Source A ATP Igt siRNADicer complex RISC siRNAs 1925 nt long SP 3 2ntoverhan s dsRNA 9 Mama SJuschi Namem 3437349 Mama SJuschi Namem 3437349 Mechanism of RNAi Step 3 minORNASI Endogenous AMA quotWequot sma RNAs 5 P pri miRNA i cytoplasm 3 exporti n5 pre miRNA I YFG mRNA 7mG qu Mei evSJuschi Aamredm 3437349 mRNA cleavage MeistevSJuschi Aamred 3437349 LL ATP microRNAs Endogenous small RNAs DICER Mersey StTuschi Nemem 3437349 microRNAs Endogenous small RNAs Translational repression Meister StTuschi Nature m 3437349 Comparison of RNAi and miRNA RNAi Viral defense Trigger long dsRNA si RNAs have internal complementarity siRNAs have complementarity to target m RNA Result in mRNA cleavage miRNA Endogenous gene regulation Trigger short hairpin RNA miRNAs have incomplete internal com plem entarity siRNAs have incomplete complementarity to target mRNA Result in translational repression siRNA design guidelines mammalian cells Antisense Guide strand Sense Passenger strand Two 2 nt 3 overhangs and less than 30 bases long to avoid interferon response typically 1925 bases long Perfect complementarity to target gene Avoid signi cant complementarity to other genes gt 10 consecutive bases Absence of internal repeats or palindromes Therm odynam ic considerations siRNA design guidelines mammalian cells Thermodynamic considerations uume strand passenger strand gul E strand ncnmnmsu mmsc 539 end unwmmng Guide strand low internal stability GC content at 5 end high internal stability GC content at 3 end Overall low to medium GC content 3052 Base preferences or A at position 1 of guide strand C or G at position 19 ofguide strand Presence of an A or U at position 10 ofguide strand Mechanism of RNAi Step 1 siRNAs 1925 nt long 5P 3 2nt overhangs Meis121amp Tuschl Name on 3437349 Approaches for RNAi 1 Synthesized siRNAs Order siRNAs Transfect siRNA into cells YFG mRNA cleavage Approaches for RNAi 2 Small hairpin RNA shRNA expression vector YFG mRNA cleavage Comparison of RNAi approaches Transfected siRNAs shRNA vectors Advantages Simpler approach Less expensive aster if homemade Can be inducible Require cloning unless ordered Disadvantages Transient Expensive Must optimize conditions Confirming and optimizing RNAi Validate RNAi knockdown Western blot RTPCR or Northern blot Controlling for offtarget effects 1 Homology to other genes a Rescue V l u MGWPNA Pomotmlmg mRNAdognhun MAM uunuumun lelNA Resistant version AAAAA quot quot1 quot RNA No pairing no silencing sscue ol phenotype Confirming and optimizing RNAi Validate RNAi knockdown Western blot RTPCR or Northern blot Controlling for offtarget effects 1 Homology to other genes a Rescue b Multiple siRNAs Tame quotIRMA WW siRNAl iRNA Z same pimncwve Confirming and optimizing RNAi Validate RNAi knockdown Western blot RTPCR or Northern blot Controlling for offtarget effects 1 Homology to other genes a Rescue b Multiple siRNAs 2 Saturating RNAi pathway a Minimal transfection dose b Control siRNA 3 Innate immune responses eg Interferon response a Control siRNA b Assay pathway proteinmRNA levels Mammalian Interferon Stimulated Genes lSGs r lemma sum RTPCR pmm R reverse Imus1 Emma prim1 nhnna Hrr nmn Mnl Ther ZEIE Resources for designing ordering siRNAs Table 1 quot I L L e siRNA sequence selection web tools Tools URLs SiDESIGN httpwwwdharmaconcom RNAi Designer BIOPREDsi httpsmaidesignerinvitrogencom rnaiexpress httpwwwbiopredsiorg Whitehead siRNA Selection server siDE siSearch httpjurawimitedubiocsiRNA httpsidebioinfoochoafibes httpsisearchcgbkise Sirna httpsfoldwadsworthorgsirnapl siRNA design software PEl amp Tusunl znne Nature Methods httpwwwcshlltuhllt sirna RNAi User s Guide Focus an RN Nature Methods Sept 2006 httpwwwnaturecomnmethfocusrnaiindexhtml Basic NMR Concepts A Guide for the Modern Laboratory Description This handout is designed to furnish you with a basic understanding of Nuclear Magnetic Resonance NMR Spectroscopy The concepts implicit and fundamental to the operation of a modern NMR spectrometer with generic illustrations where appropriate will be described It can be read without having to be in front of the spectrometer itself Some basic understanding of NMR spectroscopy is assumed IMPORTANT There is a short written test at the end of this handout which must be taken in order to obtain a NMR account This handout was prepared by Dr Daniel Holmes of Michigan State University using the NMR Basic Concepts handout from the University of Illinois s NMR service facility under the direction of Dr Vera V Mainz Her generous contribution is gratefully acknowledged February 2004 Table of Contents Basic NMR Concepts 1 Introduction 2 11 Basics of FTNMR SiX critical parameters 3 111 Applications of FTNMR 10 1 Shimming line widths and line shapes 12 2 Zero lling 17 3 Apodization 20 4 Signaltonoise measurements 22 5 Integration 25 6 Homonuclear decoupling 29 13Clt1Hgt spectra 31 1 V 8 13C1H DEPT spectra 35 IV Index 39 V NMR Basics Test 40 Introduction Nuclear Magnetic Resonance NMR is a powerful nonselective analytical tool that enables you to ascertain molecular structure including relative con guration relative and absolute concentrations and even intermolecular interactions without the destruction of the analyte Once challenging and specialized NMR techniques have become routine NMR is indeed an indispensable tool for the modern scientist Chemists with little knowledge of NMR are now able to obtain 2 or even 3dimensional spectra with a few clicks of a button Care must be taken however when using such black box approaches While the standard parameters used in the setup macros for experiments might be adequate for one sample they may be wrong for another A single incorrectly set parameter can mean the difference between getting an accurate realistic spectrum and getting a meaningless result A basic understanding of a few key aspects of NMR spectroscopy can ensure that you obtain the best results possible This guide is intended to highlight the most pertinent aspects of practical NMR spectroscopy quotAodern pulse NMR is performed exclusively in the Fourier Transform mode Of course it is useful to appreciate the advantages of the transform and particularly the spectacular results which can be achieved by applying it in more than one dimension but it is also essential to understand the limitations imposed by digital signal analysis The sampling of signals and their manipulation by computer often limit the accuracy of various measurements of frequency and amplitude and may even prevent the detection of signals altogether in certain cases These are not di icult matters to understand but they often seem rather abstract to newcomers to FT NMR Even ifyou do not intend to operate a spectrometer it is irresponsible not to acquire some familiarity with the interaction between parameters such as acquisition time and resolution or repetition rate relaxation times and signal intensity Many errors in the use of modern NMR arise because of a lack of understanding of its limitations quot From AE Derome Modem NMR Techniques for Chemistry Research 1987 Basics of FT NMR Six Critical Parameters This section will give you enough information about FTNMR experiments to avoid the most common errors We will cover the most important parameters that affect any spectrum you may collect using an FTNMR spectrometer These are 1 Spectrometer Frequency sfrq 2 Pulse Width pw 3 Acquisition Time at 4 Number of Points hp 5 Sweep Spectral Width sw 6 Recycle Delay d1 The letters in 1 39 39 r 39 Tie im 39 all Varian spectrometers The parameters are discussedin more detail below The most basic and common pulse sequence you will encounter is the 1PULSE FTNMR experiment which is the sequence used for routine 1H and 13C acquisitions It can be represented as shown in Figure 1 In a typical NMR acquisition this pulse sequence will be repeated many times in order to improve signaltonoise SN which increases as the square root of the number of scans nt The user can independently set each of the parameters shown in Figure 1 Knowledge of their purpose and function will help you obtain quality NMR spectra 0n Varian spectrometers you can view the current pulse sequence by typing dps Pulse Width pw Recycle Delay d1 Acquisition Time at Figure 1 Schematic representation of one cycle of a simple 1PULSE pulse sequence 1 Spectrometer Frequency sfrq It is called a lPULSE experiment because one radio frequency pulse pw is applied per cycle The radio frequency pulse excites the nuclei which then reradiate during the acquisition time giving an NMR signal in the form of an exponentially decaying sine wave termed freeinduction decay FID The radio pulse has a characteristic frequency called the spectrometer frequency sfrq which is dependent upon the nucleus you wish to observe and the magnetic field strength of the spectrometer NMR spectrometers are generally named for the frequency at which protons will resonate Thus a Varian Inova 500 will cause protons to resonate at approximately 500 MHz A 500 MHZ NMR Spectrometer has a field strength of 117 Tesla The spectrometer frequency defines the center of the NMR spectrum you acquire A RF pulse with an exact frequency is not desirable since NMR chemical shifts are spread out over a range of frequencies 10 ppm for 1H and N250 ppm for 13C Luckily the short pulse lengths used in FTNMR have a frequency spread due to the Heisenberg Uncertainty Principle As you shorten the pulse length and increase power uncertainty in the frequency results in a larger field of excitation A longer lower power pulse will have less frequency spread and can be used for frequency selective excitation or saturation 2 Pulse width pw Prior to applying a radio pulse a slight majority of nuclear spins are aligned parallel to the static magnetic field B0 The axis of alignment is typically designated the Zaxis and the bulk magnetization is shown as a bold a1row Figure 2 left side Application of a short radio frequency pulse at the appropriate frequency will rotate the magnetization by a specific angle 636027B1tp degrees where N275B1 is the RF field strength and tp is the time of the pulse Pulses are generally described by this angle of rotation also called ip angle The amount of rotation is dependent on the power tpwr and width of the pulse in microseconds pw Maximum signal is obtained with a 90 pulse Thus a 90 pulse width is the amount of time the pulse of energy is applied to the particular sample 90 is not 90 for all samples in order to ip all the spins into the XY plane ie the condition shown in Figure 2A The 90 pulse width for proton NMR experiments is set to about 813 us on most instruments The approximate field width of excitation is given by the formula RF eld l490 pulse Thus for a 8 us the field is l40000008 31250 Hz which is ample for the typical range of proton resonances in organic samples at 500 MHz the proton range is about 5000 to 7000 Hz The pulse width is entered in microseconds by typing pwdesired value The exact value is dependent upon the sample nucleus solvent etc as well as the instrument probe etc Methods for measuring the pulse width will be discussed in another handout and are for the most part only required for advanced experiments For routine experiments most users use a 450 pulse for their data collection Figure 2B The reasons for this are discussed under recycle delay Z Z 900 A PW 10 90 Y HSgt Y Bo X X Z Z 450 B PW5 45 Y us Y Bo X X Figure 2 The average nuclear spin magnetization bold arrow for an NMR sample placed in a magnetic eld aligned along the Zaxis before and after application of a pulse 3 Acquisition time at Thus far we have sent a pulse through the sample and ipped the magnetization by a speci c angle The nuclear spins are no longer at equilibrium and will return to equilibrium along the Zaxis In Figure l the decaying sine wave represents this process of Free Induction Decay FID which is a plot of emitted radio intensity as a function of time The time it takes to acquire the FID is called the acquisition time and is set by the parameter at A natural inclination might be to increase the acquisition time to maximize the amount of signal that is acquired Increasing the acquisition time is advantageous up to a point but will be detrimental if extended too far Care and forethought should be taken when adjusting at too long and you will acquire noise unnecessarily too short and extraneous Wiggles will occur at the base of the peaks read zerofilling section for more information 4 Number of points np The tiny analog signal emitted from the sample in microvolts is ampli ed mixed ltered and attenuated prior to digitization which is required for further computer processing The ADC analogtodigital converter converts the analog FID FT sfrq 500075 MHz gt 7 6 5 4 3 2 1 ppm Figure 3 Fourier transform of the FID for estrone acquired at 500 MHZ Note the spectrometer frequency you use in general will not be exactly 500 MHZ into a series of points along the FID curve This is the number of points np In general the more points used to de ne the FID the higher resolution The number of points np sweep width sw and acquisition time at are interrelated Changing one of these parameters will affect the other two see below 5 Sweep Width sw While the FID contains all the requisite information we desire it is in a form that we cannot readily interpret Fourier transforming the FID commonly referred to as FT or FFT for Fast Fourier Transform will produce a spectrum with the familiar intensity as a function of frequency as shown in Figure 3 The frequency domain spectrum has two important parameters associated with it the spectrometer frequency sfrq discussed earlier and the spectral width or sweep width referred to as sw see Figure 4 It is important to remember that the spectral width W is independent of the spectrometer operating frequency however since the number of Hz per ppm is dependent on the spectrometer operating frequency the spectral width M will change depending upon the spectrometer used For example at a spectrometer frequency of 300 MHz a spectral width of approximately 3000 Hz is needed to scan 10 ppm since each ppm contains 300 Hz 10 ppm x 300 Hzppm 3000 Hz At a spectrometer frequency of 500 MHz a spectral width of approximately 5000 Hz is needed to scan 10 ppm 10 ppm x 500 Hzppm 300 MHZ 500 MHZ I I I I I i II l l 10 ppm 0 ppm 10 ppm 0 ppm lt gt lt gt 3000 Hz 0 Hz 5000 Hz 0 Hz Figure 4 The spectral width in ppm and Hertz at different spectrometer frequencies Note the difference in the spectral width in Hertz for the two spectrometers The sweep width sw number of points np and the acquisition time at are related by the following equations at 1 23w and res i M 2 at np where res is the digital resolution of the spectrum The digital resolution is in units of Hzpoint and the ruleofthumb is that the digital resolution in Hertz should be less than one half the peak width at halfheight This ensures that each peak is described by at least 3 points For example if your peak width at halfheight is 05 Hz the digital resolution should be less than 025 Hz Therefore if your spectrometer frequency is 500 MHz your total spectral width is 5000 Hz 10 ppm and your required digital resolution Res is 025 Hzpoint rearranging equation 2 gives you the minimum number of points required for adequate digital resolution np M 40 000 points 3 res Since the computer works most efficiently if the number of points is a power of 2 the closest larger power of 2 would automatically be used which in this case is 65536 points The spectral width number of points and acquisition time can be speci ed when operating the spectrometer usually by typing the appropriate mnemonic followed by an equals sign and the numeric value e g np64000 The spectrometer will set the units automatically Generally Varian s automatically change the number of points according to equation 2 if the acquisition time or sweep width are changed If at np or sw are changed the data must be reacquired An alternative to changing these parameters is to use zerofilling This is described in the section titled Zero Filling 6 Recycle delay 11 On Varian s this delay time is named dl pronounced deeone and appears at the beginning of the pulse sequence see Figure 1 In practice this delay should be thought of as coming after the acquisition time It is an important parameter and plays a vital role in obtaining accurate integration After the RF pulse the nuclear spins do not instantly return to equilibrium rather they relax according to a time constant called T1 T1 is lR where R is the rate of relaxation After one T1 approximately 63 of the magnetization has returned to the Zaxis T1 s are dependent on many factors including nuclear environment temperature and solvent Carbon T1 s are typically much longer than proton Tl s Since each nucleus in a molecule is immersed in a different magnetic environment their Tl s will not be the same Not allowing enough time for relaxation between pulses will cause varied attenuation of the signals and inaccurate integration see Integration Section for more details Normally when a 900 pulse width is used to excite the spins Figure 2A atotal time TT between pulses of 5xT1 is necessary in order to have complete relaxation If a pulse width less than 900 is used the total time can be proportionally less This is why the standard pulse width for 1D 1H NMR experiments is 45 The total time between scans is given by the following equation where TT is the total time and d1 is the recycle delay TT pw at dl Since the pulse width is in microseconds while the acquisition time and recycle delay are in seconds the pulse width can be ignored leaving us with the equation TT at d1 4 The optimum recycle delay can be computed by rearranging the equation to give d1 TT at 5 As an example of the above if your longest T1 is 600 msec then the total time where TT5x T1 must be at least 3 seconds Take Home Lesson These six parameters provide the foundation on which all NMR experiments are built Appreciation of them will go far in the correct acquisition anal interpretation of your NMR spectra thus saving precious time anal effort This not only applies to simple JPULSE experiments but also is equally important in 2 D and 3 D NMR spectroscopy Applications of FTNMR 1 CHC13 Peak Width at Half Height LWlz The purpose of this section is to acquaint you with proper peak shape and the problems that are caused by improper shimming NMR peaks have a shape that is called Lorentzian A Lorentzian line can be expressed mathematically and has three parameters amplitude A width at half height in Hz LWlz and position in Hz X0 An example of a Lorentzian line with LW1z 025 Hz is shown below in Figure 5 Also NMR spectra are typically displayed as an absorption spectrum signal is as shown below as opposed to dispersive which has the signal dispersed equally above and below the baseline see Phasing section on next page f ALWl22 LWl22 4X0 X2 LW Z A Amplitude LWI2 Peak width at half height in Hz X0 Peak position in Hz Hertz Figure 5 Absorptive Lorentzian line with LW1z025 Hz The minimum obtainable peak width at halfheight is directly related to the resolution of an instrument ie how close two peaks can be and still be distinguishable Resolution is usually measured using o dichlorobenzene which has very narrow lines in its 1H NMR spectrum The manufacturers39 resolution speci cation is usually 020 Hz although peak widths of less than 010 Hz are obtainable by an expert shimmer Manufacturers of NMR instruments however have traditionally separated the resolution speci cation from the lineshape specification Line shapes for 1H NMR spectra are usually speci ed using CHC13 and the speci cations are stated in terms of the peak width at halfheight 055 and 011 height of the CHCh peak The latter two percentages are chosen because they are the height of the 13C satellites of the CHCh line and onefifth this height These values are meaningful only when compared with the halfheight width From the mathematical equation for a Lorentzian line see Figure 5 the line width at 055 height is calculated to be 135 times Lqu while the line width at 011 height is calculated to be 30 times the Lsz So if the peak width at half height is 030 Hz the calculated values are 40 Hz at 055 and 90 Hz at 011 For comparison the manufacturer39s speci cations are 1015 Hz and 2030 Hz at 055 height and 011 height respectively These values are larger than the theoretical values because the line widths at 055 and 011 height are very sensitive to shimming Other factors that in uence line shape include the quality of the NMR tube sample spinning sample concentration dissolved oxygen and paramagnetic impurities The latter three will lead to an overall broadening of the lines Phasing Due in part to delays in the pulse sequence between excitation and reception and to frequency offset errors acquired spectra will have amix of absorptive and dispersive signals Your spectmm s peaks will not look like the lorentzian in Figure 5 but have some portion that is displaced below the baseline As auser you will have to correct the spectrum by adjusting the phase of the spectrum The Plotting Practice handout will help you with phasing spectra For now it is only important to lmow that phasing the spectrum is routine and involves correcting two parameters zeroorder phase which is frequency independent and firstorder phase which is frequency dependent Conecting the phase is as simple as typing a command or doing a little bit of clickanddrag mouse work Below is an example of a poorly phased spectrum at left along with the conect spectrum ie purely absorptive peaks Not Phased Well Phased Shimming The term shimming a magnet is a piece of NMR jargon that harks back to the early days of NMR spectroscopy Originally permanent magnets were used to provide the external magnetic eld To obtain the most homogenous eld across the sample the pole faces of the magnet had to be perfectly aligned and to accomplish this small pieces of wood or shims were hammered into the magnet support so as to physically move the poles relative to each other Luckily nowadays you will not be required to bring hammer and wooden shims to the spectrometer Shimming is accomplished by changing the applied current for a set of coils surrounding the probe This applied current will create small magnetic elds in the region of your sample that will either enhance or oppose the static magnetic eld Your goal will be to adjust these coil elds by a series of mouse clicks to obtain the most homogeneous magnetic eld across your sample which is usually observed as an increase in the lock signal It is important for you to have a basic understanding of line shape so you can judge when 1 your shimming is off and 2 you need to spend more time shimming your sample The best way to avoid problems is to establish a procedure such as the one detailed below I Always load a shim library when you sit down at the instrument You should never assume the previous user left the instrument with a standard shim library loaded Without reloading standard shims you will have to start where the last person stopped and that might include someone who shimmed for a short sample a bad tube a viscous sample etc 11 Be aware of lock parameters especially if you only shim on the lock display Establish lock transmitter power and gain levels that work for most of your samples If you encounter a sample that seems to require an unusually high power or gain setting there is a problem with your sample andor the instrument and shimming on the lock level may be dif cult or impossible III Shimming problems are con rmed only if the problem is visible on m peak in your spectrum If for example only one peak is doubled the problem is sample related and can t be shimmed away Remember anomalies close to the base of intense single lines may not be visible on less intense peaks unless the vertical scale is increased IV Establish a shimming method Shimming is an art form that requires patience and practice You should always approach shimming with some method that works for you to give acceptable results Example load a shim library adjust the lock level to a maximum with Zl then Z2 then Zl then Z3 and then Zl V Spinning side bands should always be below 2 If spinning side bands are above 2 turn off the spinner air optimize the X and Y shims then turn the spinner air back on and reoptimize Z1 Z2 and Z3 If this does not solve the problem consider transferring your sample to another tube Knowledge of correct line shape can help you correct problems such as those shown in Figure 6 Although the peak in Figure 6b may have a line width at halfheight that is less than 050 Hz it is obviously poorly shimmed You should never accept a poorly shimmed line shape such as is shown in Figure 6b where a single line is expected On the pages that follow are some line shape defects and the shims that should be adjusted to correct the problem You will also notice that the FID will show the problem as well but may not be as easy to diagnose In general oddorder shims Z1 Z3 Z5 affect the line shape symmetrically while evenorder shims Z2 Z4 cause a non symmetrical line shape The higher the order Z4 is higher order than Z2 the lower closer to the base ofthe peaks the problem is observed Zl A quotgoodquot FID and a spectrum with small spinning sidebands are shown above in a btit when the quotZquot shim is changed b a characteristic beat in the FD is obtained and the spectral line broadens and manifests structure that is an indicator of 21 inhomogeneity c d 22 The asymmetric shape of the above peak 0 is typical of a misset ZZ shim Note that the beats in the FID are less pronounced than in the diagram for 21 above figure b and that the initial descent is steeper If the 22 shim is misset in the opposite direction then the asymmetry is reversed d llI II I l I h v Innn mhm mw Z3 Although the broadened base of this peak is typical of a misset Z3 shim as is the rapid initial descent of the FID there are also hints of lZl and 22 in the lineshape Z4 The very rapid initial fall of the F11 and the pronounced asymmetry are typical of a misset Z4 shim Note that in contrast to the effect of the Z3 shim effect in the previous diagram there is very little evidence of Z or 22 suggesting that the Z3 and Z4 shims windings are staggered slightly ie their origins differ Reversal of current in the shim naturally reverses the asymmetry X or Y The formation of echoes in the F11 every 50 ms is clearly visible and rstorder spinning sidebands 20 Hz away from the main spectral line can be seen Firstorder spinning sidebands are separated from the main peak by the sample spinning rate 20 Hz 715 r li39 tut I 390 Hag In MUMquot u mw XY or XzYz 1R2 Echoes are now formed every 25 ms see previous diagram and the spinning sidebands are quotsecondorderquot ie 40 Hz away from the main line which is twice the sample spinning rate The difference in the two sidebands39 heights is often seen for misset XY or XZY2 shims Figure 6 From G Chmurny and D Hoult The ancient and honorable art of Shimming Concepts in Magnetic Resonance 1990 2 131149 Shimming Take Home Lesson 16 The art of shimming resides in the fact that there is no single set of rules that work for every sample spectrometer person or even time of year Personal experience is the best and frankly only way to master shimming That being said knowledge of correct line shapes will allow you to decide quickly whether your sample is correctly shimmed M will have to decide whether the return a better line shape is worth the time spent achieving that line shape ZeroFilling As stated earlier the digital resolution is equal to acquisition time391 If you wanted to increase resolution you might consider increasing the acquisition time at to gain more points and thus better resolution This would certainly work but increasing it too much would sacri ce SignaltoNoise for the resolution enhancement The FID has a nite lifetime which is proportional to the various T1 s for a given molecule When the acquisition time is signi cantly longer than the longest T1 the contribution from noise will be quite large This combined with the increased oyerall experimental time ll llnlllllll lllllllll lllllllll lllllllll lll 2 4 6 8 10 12 14 16 18 Figure 7 Four scan acquisition of ethylbenzene on an lnoVa300 The triplet to the right was acquired with at 20 seconds the triplet at left had at 4 seconds which gave a SN twice that of the other acquisition The FID s are shown below the spectra 18 necessary to acquire a given number of scans leads to a signi cant decrease of SN Figure 7 shows the results from two separate acquisitions on the same sample with the same number of scans but with their acquisitions times differing by a factor of ve The SignaltoNoise for the 4second acquisition time at is about twice that of the 20second acquisition time and required a fourth of the time Note the FID s which clearly show that the signal decays below the level of noise around two seconds The additional acquisition time merely adds noise to the spectrum Of course there is a compromise with using a shorter acquisition time you lose digital resolution In the spectrum below there is about a factoroffour reduction in digital resolution for the shorter acquisition 015 Hzpt vs 004 Hzpt for at4 and at20 respectively Luckily there is a means of increasing digital resolution without requiring such long acquisition times This is accomplished by zero lling Zero lling is simply adding data points with zero intensity to the end of the FID This will add data points to your FID without adding additional noise It is important to note however that zero lling does not improve true resolution it only improves the apparent resolution This can be very useful because ne coupling may not be visible due to low digital resolution fn 128k W l l l l l l l l l l l l l l l l l l l l l l l l l l l l l 784 783 782 781 780 779 778 777 ppm Figure 8 Effect of zero lling on an aromatic multiplet Spectrum taken on a Varian UnityPlus 500 Spectrometer l9 even though the coupling is resolved in the time domain Figure 8 shows the effect of zerofilling on a spectrum At low resolution the ne coupling is not visible but with adding zeros to the FID the details of coupling emerge Varian executes zero lling through the Fourier number fn A Fourier transform will transform fn zeros to the nearest power of two minus np points e g if np64k and fn4np then the numbers of zeros 218 216 or 196608 points A total of 262144 points will be transformed In practice setting fn more than 4 times np is not useful One might be tempted by the preceding section to set the acquisition time at to a very short value and then use zerofilling to increase the digital resolution This will lead to spectral artifacts Figure 9 demonstrates these artifacts for the methyl triplet of ethyl benzene The spectrum on left has an acquisition time of 1 second and 4 seconds for the one to the right They both have the same number of points 200k but clearly the spectrum to the left has artifacts These artifacts are termed truncation artifacts or colloquially sinc wiggles sin XX modulation and arise from turning off the receiver before the FID has mostly decayed ll llll llll llll llll llll llll llll llll llll llll llll llll llll llll llll 15 10 5 0 5 10 15 Hz 15 10 5 0 5 10 15 Hz Figure 9 Truncation artifacts or socalled sinc wiggles because of too short acquisition time atl Both spectra have 200k points That to the right has an at4 seconds with zerofilling to 200k That to the left has atl seconds with zerofilling to 200k Spectra were taken on a Varian UnityInova 300 20 Apodization SignaltoNoise SN is very important for any spectroscopic technique NMR spectroscopy unfortunately suffers from low SN Acquiring more scans is the most straightforward if not timeconsuming means of improving SN SN increases as the square root to the number of scans ie SN nt An alternative approach is to apply a weighting function to the FID to improve SignaltoNoise Also you can apply weighting functions to improve resolution but with a concomitant loss of SN Take a Wholip ll llll llll llll llll llll llll llll ll l llll llll llll llll llll llll llll lll l llll llll llll llll llll llll llll ll 8280 8270 8260 ppm 8280 8270 8260 ppm 8280 8270 8260 ppm Figure 10 The effect of different weighting functions on an aromatic multiplet Intensities are absolute Note the differences in the FID s 21 look at Figure 10 The three sets of peaks and their corresponding FID s are from the same experiment The only difference between the peaks is the particular type of Weighting function or apodization that Was used The set in the middle had no apodization and We see an apparent doubletofdoublets J 87 and 17 Hz The SN for these peaks is 1952 the next section Will describe the measurement of SN Since SN is proportional to the initial intensity of the FID multiplying the FID by an exponential curve Wt exp1bt Where 1b is the line broadening factor should result in improved SN Indeed multiplication of the middle FID by the function Witth 2 gives the FID and spectrum on the right see companion gure 11 as Well The SN for these peaks is 87813 It is more than a fourfold improvement in the SN but at the expense of line Width We have lost the small coupling constant which is vitally 1 h vs 2 V vs 2 V vs 1174310 14e04 rm 174310 2250000 unused 174310 3619173 Figure 11 Interactive VNMR display of various apodization schemes At top are the resulting spectra middle is the Weighting function bottom is the raw FID s Le negative line broadening With Gaussian multiplication Middle no apodization Right 2 Hz line broadening important for structural elucidation Exponential multiplication imposes an arti cial mpid decay of the FID compare the middle FID to that to the right Since line width is inversely proportional to the transverse decay T2 a shorter FID means broader lines In fact exponential multiplication of this sort is termed line broadening Where 1b will be the additional line Width imposed by the function Optimal SN improvement occurs When the lb factor equals the resonances natural line width Each resonance has its own line width and therefore a single lb value will not be optimal for every peak Apodization can also be used to improve resolution by emphasizing the tail of the FID This has been done to the FID on the left of Figure 10 A function with a negative line broadening factor as well as a Gaussian function has been used see Figure 11 for the VNMR interactive weighting window which displays the function to the left This has emphasized the middle and end of the FID and has revealed an additional coupling of 06 Hz In effect it has extended the length of the signal The price to pay for this apodization is a significant decrease in SN namely from 1952 to 608 Thus you must use such weighting schemes with caution Furthermore apodization cannot make up for poor shimming or inadequate acquisition time If it is not resolved in the time domain it will not be resolved using either zerofilling or apodization SignaltoNoise Measurement The signaltonoise measurement or SN is an important criterion for accurate integrations and is also one of the best ways to determine the sensitivity of a NMR spectrometer In general a higher SN specification means that the instrument is more sensitive It is also useful in roughly determining the time requirement for an experiment Standard SN measurements for proton spectra are always determined using a sample of 01 ethylbenzene in CDC13 ETB A typical result for the Varian Inova 500 is 2001 using the 5mm probe It is important that the spectrum be acquired under the following standard conditions only for determining system performance 1 Use a 90 pulse 2 Line Broadening of 10 Hz 3 Spectral Width of 15 to 5 ppm 4 A sufficient relaxation delay at least 5xT1 5 A sufficient digital resolution less than 05 Hzpoint 6 One scan Optimum signaltonoise for any sample is achieved using a line broadening equal to the peak width at half height When this line broadening is applied the peak width at halfheight doubles ie it is the sum of the natural peak width at onehalf height plus the line broadening applied The equation used for calculating SN is SN 5 PP where A height of the chosen peak and NPp peaktopeak noise Peaktopeak noise means exactly that a measurement from the most positive to the most negative positions for the noise As shown below the widest differences are used for the measurement The distance between the two horizontal lines above in mm is the Npp value to be used in equation 5 Choice of a noise region must be consistently applied for standard samples and for 01 ethylbenzene ETB use 5 to 35 ppm SN measurement is an automated process and only requires choice of the appropriate window placement of the cursors and typing the correct command dsn on Varian s The signaltonoise of a given signal increases as the square root of the number of acquisitions therefore to double the signaltonoise you must take four times as many acquisitions When using a concentrated sample such as 57 menthol for 13C or when running routine 1H spectra the number of scans is often quite small so the point discussed above may not seem important However suppose you are in the following situation you have only a few mg of research sample and after collecting a 13C spectrum 24 for 2 hours you get peaks with an SN of only 51 Since the peaks are barely Visible above the noise and you may have missed any quaternary carbons you want to re collect the spectrum to get an SN of 501 a value more typical for carbon NMR Unfortunately this will take 10 10 2 200 hours SN Take Home Lesson At some point you may take a spectrum and wonder why the signals are so weak Over 75 of the time the problem is not with the spectrometer but with your sample You can test this quickly by taking a spectrum of a standard such as ETB or menthol In this way you can save yourself needless frustration by identifying problems that are due to a bad sample Always obtain the spectrum of a standard well characterized compound before obtaining that of your unknown Integration The purpose of this section of the handout is to show you how to obtain accurate integrals The spectrum of 01 ethylbenzene in CD2C12 is given in Figure 12 CDC13 is not used in this case because the solvent peak overlaps with the phenyl region and obscures integration If we assign an integral of 300 to the CH3 triplet then the phenyl region integrates to 412 protons while the CH2 quartet integrates to 193 protons Thus the integral for the phenyl protons is 156 too small while the integral for the CH2 quartet is off by only 35 The 142 error for the phenyl protons is not due to spectrometer error it is because we have chosen parameters for acquiring the spectrum that guarantee we will get inaccurate integrals Figure 12 1H WR spectrum of 01 ethylbenzene in CD2C12 taken on a Varian lnova 500 MHz spectrometer with no recycle delay nt4 d10 The longest T1 for this sample was measured at 12 seconds The accuracy of the integrals obtained for most routine spectra is usually about 26 1020 This accuracy is sometimes suf cient especially if you already know what the compound is However this accuracy is usually not adequate to determine the exact number of protons contributing to a given peak nor is it suf cient for quantitative applications such as kinetics experiments or assays of product mixtures where one demands an accuracy of 12 For example 20 accuracy is not suf cient to decide whether two peaks have a relative ratio of 13 or 14 Obtaining 12 accuracy can be achieved but you need to be aware of the factors that affect integrations These are as follows 111 There should be no nuclear Overhauser effect contributions or any other effects that selectively enhance certain peaks This is a problem only with X nuclei such as 13C and will be dealt with in section 4 No peaks should be close to the ends of the spectrum The spectral width should be large enough such that no peak is within 10 of the ends of the spectrum This is because the spectrometer uses lters to lter out frequencies that are outside the spectral width Unfortunately the lters also tend to decrease the intensities of peaks M the ends of the spectrum For example at 500 MHz if two peaks are separated by 7 ppm a spectral width of at least 3500 Hz is sufficient to get both peaks in the same spectrum and prevent foldovers However to avoid distortion of the integral intensities because of lter effects the spectral width should be set 10 larger on each side 350 Hz giving a total spectral width of about 4200 Hz 84ppm Thus you should be prepared to make the spectral width larger if necessary The recycle time should be at least ve T1 s Data should be collected under conditions which ensure that all the nuclei can fully relax before the next FID is taken ie if 900 pulse widths are used relaxation delays of FIVE times the longest T1 of interest are necessary In the case of 01 ethylbenzene in CDzClz the longest T1 of interest is 98 sec phenyl protons so the relaxation delay when using a 900 pulse width should be 49 seconds The spectrum should have a SN of at least 2501 for the smallest peak to be integrated Usually if you cannot see any baseline noise you probably have close to the required SN for accurate integrals V The baseline should be at Distortion due to phase problems should be corrected Baseline distortion due to nonoptimum parameter selection that causes a baseline roll Will not be discussed here See lab staff for help if you suspect this problem VI The peaks need to be sufficiently digitized as discussed earlier in this handout If the linewidth at halfheight is 1 Hz you need a digital resolution ofless than 05 Hz VII The same area should be included or excluded for all peaks For example all peak integrals should be measured 5 Hz around each peak not 20 Hz around one peak 10 Hz around a second peak etc Spinning sidebands are included in this category and should consistently be either included or excluded Figure 13 1H WR spectrum of 01 ethylbenzene in CD2C12 taken on a Varian lnova 500 MHZ spectrometer With a recycle delay of 60 seconds nt4 dl60 The longest T1 for this sample was measured at 12 seconds 28 With these points in mind let s take the 1H spectrum of ethylbenzene again The major factor for poor integration in Figure 12 was the difference in T1 s for the aromatic protons 12 seconds and the aliphatic protons N7 seconds With no recycle delay there was not enough time to allow for complete relaxation If we allow for complete relaxation by setting d1 large enough say 60 seconds then integration becomes accurate as shown in Figure 13 with only a 02 error of the aromatic protons Integration Take Home Lesson Taken from Derome p 172 The moral of this section is that there are numerous contributions to the error in a quantitative measurement made by FT NMR and while each of them may be reduced to 1 or so in a practical fashion the combined error is still likely to be significant I am always skeptical of measurements purporting to be accurate to better than a few percent overall unless they come with evidence that careful attention has been paid to the above details Homonuclear Decoupling The purpose of this section of the handout is to explain what homonuclear decoupling does Examples of a homonuclear decoupled spectrum are given in Figure 14 Homonuclear decoupling is a doubleresonance technique that uses two RF elds to affect magnetically active nuclei Homonuclear decoupling involves applying a second RF eld to cause selective saturation of nucleus A while observing all other nuclei in the Figure 14 Proton spectrum of 01 ethylbenzene in CDC13 taken on a Varian Unity 400 MHz spectrometer The lower trace is the full coupled spectrum The upper inset shows that by centering the decoupler on the triplet the quartet is collapsed to a singlet while the lower inset demonstrates the effects of irradiating the quartet molecule B C D etc If nucleus A is spincoupled to nucleus B and if the second RF eld is strong enough the result is that A is effectively prevented from spinspin interacting with B The observed B nucleus spectrum will appear as if it is not coupled to 30 A The A resonance commonly appears as a glitch as a result of this experiment As shown in Figure 14 if the triplet is homodecoupled the quartet collapses to a singlet Similarly if the quartet is homodecoupled the triplet collapses to a singlet You may recall that a relatively high power short RF pulse will have a frequency spread due to the Heisenberg Uncertainty Principle therefore the second RF eld used for the selective decoupling will be lower power and have a longer duration Homonuclear Decoupling Take Home Lesson H omonuclear decoupling is a fast and e ective way to establish that two nuclei are spin scalar 11 coupled and can be used to simplify a complex coupling pattern for further analysis It is also useful as a follow up to a COSY experiment to con rm specific couplings To obtain de nitive data the two signals must be separated by at least 05 ppm It is also important to note that other signals close to the irradiation point may experience a displacement in their chemical shift due to the decoupling eld This displacement in the chemical shift is called a Bloch Siegert shift and can be used to measure the decoupling field strength Proton Decoupled 13C NMR spectra 13C1H The purpose of this section of the handout is to give you some useful information about 13C lH NlVlR spectroscopy Since only about 1 in 100 carbon nuclei are NlVIR active 110 are the NMR active 13C isotope any means to improve SN is essential Splitting of the 13C resonances as a result of coupling to attached protons will result in decreased SN and is thus undesirable Therefore 13C NlVlR spectra are typically run proton decoupled The symbol 13C lH is used to denote this and implies the 13C nucleus is observed while the proton nuclei are being irradiated thus decoupling them from the 13C nuclei A typical 13C lH spectrum 57 menthol in acetoned6 is shown in Figure 15 200 180 160 140 120 100 80 6O 40 ppm Figure 15 A 13C lH NMR spectrum of a 57 solution of menthol in acetoned6 acquired on a Varian Unity 400 MHZ spectrometer This is a double resonance experiment with the observed nucleus 13C and 32 decoupled nucleus H on separate channels This experiment is called heteronuclear decoupling and is a lPULSE experiment as described in the Basics section With the addition of a decoupling field as shown in Figure 16 It is the heteronuclear version of the homodecoupling experiment With the exception that broadband saturation as opposed 45u 115 pw U to selective saturation is used vnmrseq11b52pul Figure 16 Representation of 13C1H1PULSE NMR experiment as presented on a Varian Inova 500 MHz Spectrometer Note that the pl pulse is unused in this sequence When acquiring spectra of nuclei other than protons so called X nuclei it is important to remember the following considerations 1 The Nuclear Overhauser Enhancement The 13ClH lHR spectrum obtained using a standard lPUL SE experiment is not quantitative ie the integration of the peaks Will not give a true indication of relative ratios because of a phenomenon called nuclear Overhauser enhancement NOE arising from the continuous broadband saturation of the protons 13C nuclei that have directly bonded protons can exhibit a signal enhancement of up to 198 198 or an almost threefold improvement in signaltonoise The NOE is from the dipolar throughspace coupling of the carbon and proton nuclei and is dependent on many factors Thus the NOE will be different for each unique carbon in a molecule To obtain quantitative 13ClH spectra you must do two things follow the protocol given earlier on integration and carry out a gated decoupling experiment in which the decoupler is gated on turned on during the acquisition time and gated off turned off during the recycle delay This is shown in Figure 17 105 00u Tx d1 p1 d2 pw A B C vnmrseqlib52pul Figure 17 A gated decoupling pulse sequence for 13ClH acquisition that has no nOe enhancement Note that the decoupler channel Dec is only on during segment C which is the pulse and acquisition time Compare to Figure 11 The result of this experiment is a 13CIH spectrum without NOE and is necessary for obtaining quantitative 13C spectra 34 11 T1 relaxation times The Tl s of 13C nuclei are in general longer than those found for protons as shown below in Figure 18 Therefore you may have to wait very long times if you want accurate integrals from spectra For example from Figure 18 quantitative integration of ethylbenzene would require a total acquisition time TT of 536 seconds or 3 minutes per scan A paramagnetic relaxation agent such as Cracac available from Aldrich can be used to shorten the Tl s but can sometimes be difficult to separate from the compound Note that the quaternary carbons have considerably longer T1 s and as a result typically have much smaller signals than other carbons 14 7 16 CH239CH3 CH3 N02 36 38 56 13 20 69 87 66 57 48 CH3 CH2 CH2 CH22 13 21 69 9 15 49 Figure 18 Examples of some representative 13C NMR T1 values in seconds 13C NMR Take Home Lesson Obtaining useful 13C 1H spectra requires knowledge of the same basics as needed for obtaining useful 1H spectra When your spectrum doesn t look right you can save frustration on the instrument be taking a quick spectrum of a 13C standard and checking the SN or seeing if the standard is decoupled properly 13CIH DEPT Spectra Distortionless Enhancement by Polarization Transfer DEPT is an experiment that utilizes a polarization transfer from one nucleus to another usually proton to carbon or other X nucleus to increase the signal strength of the X nucleus DEPT is an example of multipulse multichannel experiment which uses synchronous pulses on two channels to afford polarization transfer The pulse sequence is shown in Figure 19 In this case the decoupler channel Dec is proton and the observe channel is carbon Since we are transferring the population difference of the protons to the X nucleus and gaining signal from these protons it is the proton T1 s that are important in determining repetition 90u 90u 180u 90u 205 35m 35m Tx d1 jtau l 5pp rof2 rof1 pw 2 0pw pw pw jtau 2 0pw 1 Oe 1on jtau pp Z 0pw rof2 2 1e 5 342u 684u 171u U m n f s pp 2 0pp multpp A B C vnmrseqlibdept Figure 19 Schematic representation of a DEPT pulse sequence as displayed by a Varian Inova 500 MHz spectrometer The observe Tx channel is carbon and the decoupler Dec channel is proton rate for a DEPT experiment Proton T1 s can be significantly shorter than that of carbon and especially short compared to 15N and 29Si which allows you to acquire more scans 36 per unit time than the XIH experiment and thus obtain improved SN A further advantage of this population transfer is the ability to perform multiplicity editing By varying the length of the last proton pulse multpp from 45 to 1350 degrees the multiplicity of the carbon or X nucleus can be determined ie depending on the pulse the signal for a methine methylene or methyl will either be a positive negative or null signal see table below Remember since quaternary carbons have no attached protons they will show no signal Also the signal from the deuterated solvent will be absent An example of a DEPT135 experiment is shown in Figure 20 Compare it to Figure 15 In general you can run DEPT on most samples without additional calibration 7o 65 60 55 50 45 4o 35 3o 25 20 15 ppm Figure 20 An example of a multiplicityedited spectrum Expansion of a 13C1H DEPT135 spectrum of menthol taken on a Varian Unity 400 MHz spectrometer The relative sign of the peaks shows the multiplicity of the carbons The negative peaks are methylene carbons while the positive peaks are either methyl or methine carbons If you obtain less than favorable results calibration of the polarization pulse pp on the Decoupler channel can be performed This is typically done using a DEPT90 arraying 37 pp and looking for a maximum in the methine signal without contributions from other carbons Relative Intensities from DEPT Pulse Angle C quaternary CH methine CH2 methylene CH3 methyl 45 0 0707 1 106 90 0 l 0 0 135 0 0707 1 106 DEPT Take Home Lesson DEPT is an e ective means of determining 13C multiplicity that when combined with other NMR spectra anal other experimental techniques MS F T IR etc can be an invaluable tool for the analysis of unknown compounds Index A Acquisition Time at 2 3 4 5 7 8 9 18 19 20 23 34 35 40 Analogitoidigital converter ADC 6 Apodization l 21 22 23 41 B Basics of FT NMR Six Critical Parameters l 3 C Carbon NMR 32 35 Carbon NMR factors affecting 33 Carbon NMR pulse sequence 33 Carbon NMR quantitative 34 D DEPT 2 36 37 38 DEPT 1357 37 DEPT relative intensities 38 Digital Resolution res 8 l8 19 20 23 28 40 F Fourier Transform 2 6 7 20 Freeiinduction decay FID 4 5 6 7 l4 l8 19 20 21 22 23 27 40 41 H Homonuclear Decoupling 30 31 I Integration 1 9 26 29 33 35 42 Integration factors affecting 27 Introduction 1 L LinerShape 1 11 13 14 17 N Nuclear Overhauser Enhancement NOE 33 34 Number of Points np 3 6 7 8 20 P Peak Width at Half Height 11 12 Pulse Width pw 3 4 9 27 Quiz NMR 2 40 R Recycle Delay d1 3 5 8 9 26 28 29 34 S Shimming 1 13 14 16 41 Shimming Figure 16 SignaletoeNoise SN 3 18 19 21 22 23 24 25 27 32 34 35 37 41 SignalitorNoise SN calculation 24 Spectral Width sw 3 7 8 2740 Spectrometer Frequency 3 4 6 7 8 Spectrum Phase ll 12 2841 T Tl longitudinal time constant 9 18 26 28 2935 36 Table of Contents 1 Truncation Artifacts 20 W Weighting 1 21 22 2341 Weighting Line Broadening 22 23 24 Z ZeroeFilling 1 6 8 18 19 20 23 NMR Basics Test 1 Diagram and label the three parts of the lPULSE FT NMR experiment 2 What is the result when you apply FT to a FID time domain signal 3 What is the relationship between numbers of points spectral width acquisition time and digital resolution What is the ruleofthumb for adequate digital resolution Which of these parameters would you change if you wanted better digital resolution and why 4 What is the peak shape found in most solution NMR spectra 5 What shims should be adjusted if the peak shape is asymmetrically distorted 6 What is the single best factor to tell Whether a sample is poorly shimmed 7 Match the spectral artifact With its cause draw a line from the methyl triplet to the cause A i l M J39 WM km MAMJ i J w in LR I I I I I I I IIIIIllIIIIIIIIIIIIIH IIIIIIIIIIIIIIII I I I I I I I I I I I I I I I I I I IINIIIIIIIII IIIIII W W 126 123 12oppm 132 129 126 ppm 126 123 120 ppm 132 129 126 ppm 1 31 1 28 ppm I AMAV Improper Phasing Good Spectrum Low Resolution Poor Shimming FID Truncation 8 What type of apodization would you use to improve signaltonoise SN What is the disadvantage 9 Given that after 100 scans 5 minutes the SN for a sample is 35 1 how long Will it take to achieve a SN of 3501 41 10 What are the siX factors that can affect the accuracy of a 1H NMR spectral integration Why Are there any additional factors that affect the accuracy of 13C 1H integration Why 11 Is there a difference between the lPULSE FT NMR experiment used to acquire 13C IH NMR spectra and that used to acquire proton spectra If yes what is the difference 12 For a DEPT135 spectrum what are the relative intensities for the different multiplicities of carbon 10 NMR Hardware In this lecture we Will begin to familiarise ourselves With the more practical aspects of NMR ie major components of a spectrometer With probeheads and With the de nition of signal to noise 101 The spectrometer A spectrometer consists of the following components shim power uppl roe guuonpuoomdns peeqeqmd pulse programmer l H amplifier lH frequency 47 Li 1 13C amplifier w 7 7 13C frequency i receiv er computer i The magnet The magnet which provides the go eld needed for precession to take place is a superconducting magnet This means it is made out of superconducting material eg Bi Sr Ca Cu O based superconductors that has a minimal resistance at UK 27315 00 These temperatures can be achieved by immersing the superconducting material in liquid helium The superconducting coil is made out of Wire Which is several miles long and wound into a multi turn solenoid The Wire itself is made out of different materials arranged as illustrated below The reason for this arrangement is to minimize the chances of a quench if there should be material failure in the coil Superconducting MateIial Insulating Material To keep the coil cold it is immersed in a dewar containing liquid helium and kept at 42 K Some magnets have a pump built in so that the helium can be supercooled This is used to reduce the resistance in the coil further allowing higher B0 elds to be achieved In order to slow down the evaporation of helium the dewar is surrounded by dewars of nitrogen as shown in the diagram below Liquid Helium Liquid Nitrogen Container amp Support Superconducting Coil IDlIII Reflective Mylar Vacuum acuum He Reservoir Superconducting Solenoid ref httpwwwcisriLeduhLbooksnmrChapi7Chapi7htm Ghe outer vacuum region is lled with many layers 03 re extive mylar lm which serves to insulate the magnet by diminishing the amount of heat which can enter the helium region ii The shim oils The shim coils which are situated inside the bore of the magnet provide compensatory magnetic elds such that the net go eld is spatially homogeneous Inhomogeneities in the go eld arise from the way in which the magnet is designed from materials in the probe eg metallic objects in proximity to the RF coil in the probe from the sample tube from sample permeability and from ferromagnetic materials around the magnet As a result of the inhomogeneities a eld gradient may eXist across the sample eg k l Br6 gtBr5gtBr1 r6 a Higher Field r7 5 CH31 CH32 and CH33 all have different l4 a frequencies giving a broad signal rs f A 2 r2 in L F39Id rI owerle 100 50 0 Properly adjusting the currents in the shims will fillin the inhomogeneities hopefully yielding a homogeneous field and sharp symmetric peak lineshapes Z1Shim ZZShim is r5l ill394E i The shim coils consist of Wire through Which current is passed so that small magnetic elds are generated These coils have particular shapes Which correspond to particular functional forms Some typical shirn wils and their corresponding functional forms are Shim Field Shim Field Shim Field zo zl ref httpwwwcisnLeduhtbooksnmrchap chap77htm Shimming consists thus in nding the optimal current settings such that the 30 eld is homogeneous This can be determined by either maximizing the lock signal see below or by maximizing the size of the free induction decay FID of for example water Because the lineWidths observed in the solid state are typically broader than in solution state it is not necessary to shim very precisely This is often Why most people only optimize the lower order shims Z0 Zl Z2 X XZ Y and YZ and do not modify the iii The lock shim stack LockReference Freqrency LockReference Frelzuency 5 higher order shims eg Z4Z5XZ2XZ3 in the magnetic eld eg from a tram metallic trolleys In solution state it is very common and works by using the signals arising from deuterated the computer The signal is locked on to the frequency of something like D20 and monitored constantly during the course of the experiment Small changes are compensated for by a coil in the LockDisplay on a Varian Instrument When Locked On Resonance Lock Display on a Varian Instrument when field is offset 20 needs to be changed W The lock is needed to compensate for external drifts solvents in the sample which are then monitored by ln solid state the lock isn t as commonly used but can be useful as illustrated here for a 130 line of adarnantane spinning at 4 kHz For some solids probe assernblies the lock is situated in the duct used to eject the MAS rotors The deuteriurn sample in this case sits close to the RF coil but is by it iv The probehead The probehead contains an RF coil Which produces the BEG magnetic eld needed to perturb the spins away from equilibrium The major component Within the probe is a resonance circuit built from inductors and capacitors which allow the probe to be tuned and matched to a particular frequency Larmor frequency of the nuclei to be observed andor excited We saw in Chapter 2 that a probe consists of a slightly more complex Version of an LC circuit In our circuit we said last time that the inductor L is the coil where the sample is placed There are a number of different coil geometries which are used In solution standard coils are the saddle coil Saddle Coil and Helmholtz coils Magneutheidlmasfm Helmhclu tolls Whereas in solids a solenoid or at coil is used The choice on geometry depends on power applied to the coil lling factor and homogeneity V The frequency generator The frequency generator also known as the synthesizer generates a sinusoidal modulation which has a frequency close to the Larmor frequency of the nucleus to be excited perturbed The base frequency generated is called the carrier frequency or reference frequency such that the synthesizer output is de ned as 8 c08wreft l t 101 Where t is the phase of the radiofrequency modulation Vi The pulse programmer The pulse programmer interprets the pulse program that the user writes It controls how long a pulse is on for ie gating the phase of the pulse and the shape of the pulse eg rectangular Gaussian k Non ngShifted wave gb Shift instruction Gate instruction gt MH Levitt s book p 74 75 Vii The ampli ers The ampli ers increase the amplitude of the output of the frequency generator so that the nal amplitude of the RF pulses ranges from milliwatts no ampli cation to kilowatts The output of an ampli er is characterised by its peak to peak voltage Vpp or power in Watts P ie W292 400 Where Vpp is the voltage measured on the oscilloscope End dB is the attenuation used a U P gtilt101 C 102 Just as a probehead can be tuned to a speci c frequency so can an ampli er To tune an ampli er the following procedure can be used 1 Connect the output of the ampli er into an attenuator eg 40dB The output of that can now be connected to an oscilloscope for observation 2 Start pulsing using a single pulse With an easy to observe pulse Width eg 1 2ms Use a power level setting Which is in the middle of the available range WARNING Beware of connecting high power outputs directly to the oscilloscope 3 Most ampli ers have a button for tuning and one for the ampli er load Change the tuning and load so that the maximum amplitude largest Vpp value is achieved vii Filters Although the probehead behaves as a lter for a particular frequency that is to be excited and or Qbserved there are often spurious frequencies still 0L Lowpass lter cane mam l 1 Bandpass lter a present in the lines leading up to the receiver To eliminate these unwanted frequencies a number of lters can be used namely low pass lters high pass lters band pass lters or band stop lters A low pass lter allows low frequency signals to be transmitted While attenuating higher frequencies A high pass lter behaves in an opposite manner in that it lets through high frequencies While attenuating low frequencies A band pass or band stop do not attenuate or attenuate a range of frequencies respectively Schematically dB nghpass lter u n 9 9 Bandslop ller Kef H Ludwig and P Bretchko RF Circuit Design Theory and Applications Prentice Hall NJ 2000 A low pass lter consists of a circuit containing a resistor R and a capacitor C A high pass lter consists of a circuit containing a resistor R and an inductor L And a bandpass lter consists of an RLC circuit For NMR purposes the best is always to use a bandpass lter since only the frequencies to be excited observed are let through These lters are more expensive though and so are often replaced by a low frequency lter or a high frequency lter viii The receiver As With a radio a NMR spectrometer is equipped With a receiver to detect the RF or magnetization coming from the sample in the probehead Typically it consists of a quadrature detector Which is a device Which separates the Mm component of the magnetization from the ZMy component Electronically this is done With a doubly balanced mixer For more details cf R Ludwig and P kBretchko RF Circuit Design Theory and applications Prentice Hall NJ 2000 Co axial cables Connecting all the different components of a spectrometer are co aXial cables These cables consist of H an inner cylindrical conductor of radius a N surrounded by a dielectric material such as polystyrene polyethylene or te on OJ an outer conductor Which is grounded to minimize radiation loss and eld interference H and an outer insulator kTo avoid losses between the different components of j Ge spectrometer it is important to use good quality co aXial cable usually the thicker kind X The computer The computer controls most of the components listed above and acts as an interface between the user and the hardware 102 Signalto Noise Ratio When we discussed probes in Chapter 2 we introduced the concept of the sensitivity of the probehead A measure of this sensitivity in terms of the electronics is given by the quality factor Q There is another measure of sensitivity known as the signal to noise ratio given in terms of the observed magnetization In a NMR experiment the signal is given by S olt w0M O 103 where wow is the Larmor frequency and Mg is the nagnetization detected in the receiver Ghe noise known as thermal or Johnson noise coming from the electronics is N 0C kefquwo m 104 With kre f by is a proportionality constant Which takes into account the Q of the circuit and other electronic parameters of the detection devices Therefore the signal to noise ratio is given by S N keff mWQ QCMQCTQ QC 105 Where Tg m is the transverse relaxation time Using Curie s law for the magnetization for a spin 12 Nm7m2h2BO Mm 106 427T2k3T the signal to noise ratio can be rewritten as E keme fWBOTN 10 7 k N 427T2k3T Ghis latter equation shows that one can expect improved sensitivity if 1 the probe has a high Q keffw is high 2 there is a lot of sample N96 large 3 the spins observed have a high 7 value 4 the magnetic eld is large eg 800 MHZ vs 400 MHZ 5 the temperature at Which the experiment is carried out is low Nuclear Magnetic Resonance Spectroscopy Ryan Marcheschi marcheschiwiscedu 10132008 NMR Lecture Outline Lecture 1 101308 r Introduction to principles and applications of NMR r1D NMR rMultidimensional NMR rRealWorld Examples of NMR Lecture 2 101508 NMR websites wwwnmrfamwiscedu r Nuts and Bolts NMR hardware wwwbmmwiscedu 39 Tou r N M R wwwcisriteduhtbooksnmrinsidehtm wwwcemmsuedureuschVirtualTextSpectrpy nmrnmr1htm httpnobelkaistackrshbaedocnmrdochtml httpteachingshuacukhwbchemistrytutorialsm olspecnmr1htm wwwchemwisceduareasreichHandoutsnmrh hdatahtm What is NMR A Brief History Isidor Rabi Edward Purcell Felix Bloch 1944 A 1952 Described Nuclear Resonance First observed NMR in solids and liquids Richard Ernst Kurt wuthrich Original Varian A60 Spectrometer 900 MHz Varian What can NMR be used for rStructure Determination rNMR Magnetic Resonance Imaging rMacromolecular small molecule interactions rEnzymatic Analysis rMetabolomics 39 Etc NMR Topics Nuclear Spin M rResonance Frequency rNuclear Shielding quot1 rChemical Shift rCoupling 7 rMagnetization Transfer rExchange Regimes r1D NMR r2D multiD NMR Nuclear Spin rAtomic nuclei have nuclear spin 1H 13C 15N 17C 19F 31F 338 rSpin angular momentum I produces a magnetic Bo moment u rPrecession frequency is known as Larmor frequency resonance frequency
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