MOLEC STRU DYNAM NMR
MOLEC STRU DYNAM NMR BCH 6745
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This 22 page Class Notes was uploaded by Kavon Huel on Friday September 18, 2015. The Class Notes belongs to BCH 6745 at University of Florida taught by Arthur Edison in Fall. Since its upload, it has received 11 views. For similar materials see /class/206960/bch-6745-university-of-florida in Biochemistry and Molecular Biology at University of Florida.
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Date Created: 09/18/15
Today s Lecture 11 Fri Oct 27 2D NMR Review and Summary a b NOESY c Other Important biological experiments d Heteronuclear correlations lson Univasity ofFlorida 2D Exchange NMR Milli umuoads Aouonbol qz 9M3 Him 1 u m 2D NMR WWWW Preparation Evolution Mixing Detection Nuclear Overhauser Effect J Saturation around 13 ppm eg methyl 6 i i i i i W W n 55 75 7 ss sn 45 Au 35 3D 2 s 211 is m Saturation around 45 ppm eg no saturation I 75 7m 5s 5 ss sn 45 Au 35 3D 2s 2D 15 Ethyl Benzene NOE Difference spectra ppm uhHiLhruunan in mu 1 i i 1 A 75 7n 65 an 55 5 5 6U 35 3 ppm an 75 7 65 an 55 5 5 on 35 an 25 2 15 1D ppm Ethyl Benzene NOE Difference spectra Difference p m saturate CHj Difference spectrum saturate CH2 Qualitative Description ofthe NOE The lllenual hmnllhnum WWW difference in energy between ms re and ms tw e the spectrometerfrequency Cm Rclnxnliolt tmmnhcqulhhmnl a exerte thattxansmon erlramsorr NOE IzNaaeN aNa eN II x SzNaaeNa N a eN VD ttmsrmmxmmn mum A39 1 w crass nlurmruu acme Qualitative Description of the NOE 0015 0015 The NOE only can measure distances up to about 5 A because the effect depends 1 r6 mm on r396 Where r is the distance between the mm two interacting protons 0 0 3 4 5 6 I Heteronuclear NMR 90X 1 A gt X S A U lson Unwasity ofFlorida HSQC or HMQC correlate 1H and 15N Each amide group in a protein gives rise to a peak in a 2D spectrum 15NHSQC of 1A3 an unfolded protein 127 HMQC Heteronuclear Multiple Quantum Correlation 90x 180x 1 H A A P 90x 90x V s A H 22 22 H A Homework Do the product operators for both a single I spin no S coupling and an 18 coupled pair Due Wed next Wee A S Edison Univasity ofFlorida Next Lecture 13 Mon Oct 30 Assignments of peptides proteins and small molecules A S Edison Univasity ofFlorida Molecular Structure and Dynamics y NMR Spectroscopy ECH 6745c and ECH susL Fall 2006 nstrumurs Arthur 5 Emsun and Juarma Lung emaH address amm m edu ampjrungmb m edu omce H3487 m1 Humme Mcanngram nsmute Web page mm mass utes mm mm Huurs By appmntment Recommended Materials by Ray FrEErnan A 5 mm A 5 mm Umusny a M musqu mm 2m 2m Today 5 Lecture 1 Fnday Nov 29 Behaviur nf nuclear spins in a magnetic fieldI Sta anerlach Improved Steererlach BnefAngular momentum remw Rabbx expenmem gym2pm L919 A 5 mm 211 Energy Levels A 5 mm Umvusny x mm Lhmmxyai mm m m Smererlzch Experiment Anypanil mmmm u 0 am y uni Lg 7A a 1H we we 3 u o gt gt gt we we I Impruved smraneriach Experiment Feynman Lectures un Physics Impruved Steererlach Experiment Feynman Lecmres an Physics gin panik g 0 athinpanilz eg uxmm 92 eagxilluxmnu SB 0 neewe have seieetea apure E cumpunmt aluug the zraxxs it stays in that state a s mm a s 211mm Urimsqu Lhmsuyaimrma ma ant Impruved smraneriach Experiment Impruved Steranerlach Experiment Feynman Lectures un Physics Feynman Lecmres un Physics back 7 uut n panik athinpar tlz mg n39llu nlm Lgn39llu nnu a s FAmm Umu39sny a rim am Whatever happmsd alung the 17 axis duesn39tmatter mymureif we luuk ting the Kraxis itis ee again sphtintu z heams a s FAmm thwis yaf Hand zans What is spin SW 1 way uf swing is umpmmag and39hesquare arm magntude Laneahmly may the same elgan nthans 21er Wn An What is spin When apamclelsmstau 1 we can lmww the a campmem I I h211 1 More Speci cally A my imam 25mg whamme calla hyquotzrld dwnquot lard 2 mlquot mi Mtge Wewl nsu ynlim B um a e x mm hm 142 la e Mammal mam E 39 a b m sum Mayan degemn n la abseme magch rail IIa 2II1a II h2111 Tammi m i mhim hul ngnnude uflllz anguhr mnmmlum Graphical lnterpremtinn To Summarize Value ufthe angu ar memehmm along the zraxls A s Edison Umu39sny A W m Spin angular momentum is proportional to the magnetic momen he magnehcmoment pi is avectorparallel e s lar memehmm The gymma rmagietugy m ratio y is A P hysical constant pamculaxtu A given nucleus Now we can nd the energy of a magnetic moment in a magnetic eld Eu39B The magmatic eld B is also avectur The he pheauei ufz veems e g Ii and B is A scalar 1h NMR we stztvnth alarge v ueefhshihg ezraxl pl EM is the heme ufthe energy for apartmlar value ufthe quantum numba m A s Edison Umu39sny A W m The SterneGerlach experiment can now be understood h e eh Apameie with Amsghe e memehiih Amsghehe eld is phepemehsi h the denvanve gadient ofthemagneti m eld in the direeuuh ufthe force Nu gradienL he force A s Edison when mini zhhi I I Rabi mulecular beam experiment to measure 1 Feynman Lectures on Physics Bu The null pmduces a maywn Frequency GamerElm new alung me xans gumg mm me buard m When the sequency reaches re nance parades nu lungerreach the detectur The Boltzmann equation tells us the population 0 a 5 ate il we know its ener y m a 5211mm umwnma m Next Monday s Lecture 2 Man Oct 2 Behavinr nfnllclmr spins in a magnaic eld H a Teach 5pmquot apparatus b Blush equauuns e FhenummulugcallntmducnuntuTl ande 2 RF Pulses a 5211mm Umu39sny a Flaxm ma Today s Lecture 3 Wed Oct 4 Introduction to NMR Parameters a Review of Bloch equations b Chemical shi iBMRB database c J couplingiKarplus equation d Tl e T2 A S Edison Unwasity ofFlonda 2006 Bloch Equations yaxis pulse xaxis pulse In the Bloch equations magnetic elds along the x andy axes create B1 elds or v pulses These are typically yams pulse applied for short durations and the length of time the pulse is turned on is adjusted to give a desired rotation such as 90 or 180 degrees xaxis pulse A S Edison Unwasity ofFlonda 2006 RF Pulses M20quot dz meBm yManrlto w P J P 1 yaxis pulse xaxis pulse In our simulation of the Bloch equations we showed that for protons F2n267107 HzT and a B1 eld of 93quot10395 T it takes 40us to rotate the magnetization one time around the X or y axis The relationship between rf eld strength and pulse length is simply lson Unwasity ofFlonda 2006 What range of frequencies can be covered with a given rf pulse 9quot i 1 B1 211 PVT360quot I This relationship can also be understood from the Heisenberg Uncertainty Principle l This says that a 40 us 3600 pulse will create an effective eld strength BlZn of25000 Hz This tells us that we can only know the frequency of a 40 us pulse to within about 25000 Hz Thus ALL frequencies in this range are effected but not all equallyl lson Unwasity ofFlonda 2006 What range of frequencies can be covered with a given rf pulse Since a wide range of frequencies can be covered by a pulse do they all behave the same NO A frequency Am from the center carrier rf frequency will actually experience an effective eld strength given by The rotation axis will also be tilted at the angle p This can cause problems However if IE1 is much larger than A u the problems are minimal This is 1131 why we like short pulses lson Unwasity ofFlonda 2006 A 90 degree pulse followed by turning on the detector leads to a Free Induction Decay FlD The signal decays Beats with Regular spa quot lilillllllllllllnum A lllullllllnlquotquot391quotvan Different decay rates can be seen 13C NMR FID of Methyl ccDArabinofuranoside in CD3CN Collected at 117 T by Jim Rocca in AMRIS U1 U2 U3 U4 U5 IE U7 IE IS 1E 11 12 13 14 15 16 17 1B 1955 A S Edison Unwasity ofFlonda 2006 nnz Expansion of previous FID Most of the signal in the fast relaxing peak is gone by around 100 ms Peak to peak spacing is just under 50 ms 1 Now we can see complicated ne structure EIEIA DUE DUE EllEl D12 D14 D16 D18 DZEI A S Edison Unwasity ofFlonda 2006 Fourier Transform of previous FID Some peaks are bigger than others CD3CN The FID had several different frequencies These are called chemical shifts This peak has other stuff going on l lson Unwasity ofFlonda 2006 Expansion of big peaks The width ofthis peak is about 10 Hz This scalar J coupling k k 1ng or my The indicates that It IS Thls J lsjust over The width of the natural linewidth experimental sources these peaks IS ofinhomogeneities This comes from the 20 HZ z rapidly relaxing part of the FID They come from the slowl relaxing part of e FED 39InImiiumiluwwiI m The value for the J coupling is the inverse of the spacing between beats on the FID 120 MO 100 90 80 70 60 50 40 30 20 Wivasity p F londa 2006 Hz vs ppm This equation which should have already established an important location in your brain tells us that the frequency in HZ m0 of a particular resonance is linearly related to the magnetic eld strength B0 PROBLEM A given molecule has di erent values of no for different magnetic elds How can we compare results at different eld strengths ANSWER Normalize the results to make them independent of magnetic eld The value of a chemical shift in HZ referenced to some standard value is divided by the base spectrometer frequency in MHZ The units of this conversion are HZ or parts per million Because no yBois linear 1H data at 500 MHZ 267lO72n HZT 1172 T has the same ppm value at any other eld strength Forexarnple two chemicalshifts separated by39l ppm on 6 OOLMHZ magnet have 600 HZ between them Thesarne twoshi s at 500 MHZ arelstill 1 ppm apart but are only separated by 500 HZ A S Edison Univasity ofFlonda 2006 Chemical Shifts Chemical shi s are in uenced by the electronic environment Therefore they are Linle n ru pnnenrn 39 F1851 m nu mm or at 739 h a 30 u 139 unmet Table from N A 5 ram httpwwwcern msuedu reuschOrgPagenmr htm Ummmmm m Chemical Shifts Chemical 39 39 39 39 39 hmquot 9 man I ey I L 39 L we C u nu u u um NMIRis the BioMagRes data Bank BMRB at University ofWrsconsin A s Edlsan UnwexsnyafFlnndA 2mm Chemical Shift Index CS1 r h t A use each h olhas a dlfferent basequot chemlcal ehmohmeht For example an l alh alahlhe 15 dlffelent from an 1Hm glyolhe Here 15 apamal table that allows for the detexmlnatlon ofthe csI Complete oletalls ahol aolohtlohal references can be fouhollh the reference below These are average chemical shifts in certain nuclei in ammo Wlshan and Sykes chemleal shl s as a tool fur structure detemhhatlohquot Methodslh Enzymnlngy 239pp 3637392 1994 A s thseh Unwexs yanlnnda zoos J coupling and chemical shift example All ofthe whiting are 1 Enuplmgs uuuuuuuuuuuuuunuuu 1H N39MR 1D spectra ofMethyl arDrAIabmofumnosldlI m CD3CN Collected at ll 7 T by Jlm Rocca lh AMIRIS A 5 MM Unwexsnyanlnnda zoos J coupling JJL l l 1 coupling all owsyou to identify atoms within 3 sometimes more covalent chemical bonds This will be even more poweiful when we get to 2D NMR expenments Decouple 4 05 ppm W Decouple 4 93 ppm W Alumniml A s Edlsnn Umnityarplnnda zoos Why are J coupling values different Martin Karplus showed that from H atoms depends pi tons This relationship can be approximated by the famous Kaiplus equation s 19 19 Acnse tgcnsw c A B and c are empincally denved parameters I J couplings provide an estimation of molecular s Edlsnn Umnityarplnnda zoos T1 and T2 T1 is the time constant for magnetization to relax along the zaxis R111quot1 is the rate constant for the same phenomena T2 and R2 are the corresponding constants for relaxation in the xy plane Why are they important We have seen that chemical shi s 5 are in uenced by the electronic environment of the molecule Because of munBU it is clear that the magnetic field must be different for different chemical shi s The electronic environments create these different magnetic elds When molecules move either as a rigid rotor or from internal motions these same electronic environments can also act like rfpulses with one big difference rfpulses are coherent andmolecular motions are incoherent The difference is important andis essentially the difference between a grou effort where everything happens together and in a controlled manner coherent vs individuals doing their own thing randomly and whenever they want incoherent The incoherent motions cause transitions between states which lead to relaxation Quantitative details onT1 and T2 will be given in a later lecture A S Edison Univasity ofFlonda 2006 Homework for Friday In a 1H NMR experiment the carrier frequency is typically placed in the center of the spectrum T his coincidentally corresponds pretty closely to water in aqueous samples For a particular NMR experiment at 1 17T 500 MHZ the carrier frequency was placed at 48 ppm and a 90 degree pulse length was found to be 8 pus Calculate the following a What is the rf eld strength yBlZn on resonance b What is the effective eld strength at 12 ppm c Relative to the axis ofthe applied B1 eld what is the angle ofthe axis of rotation for the resonance at 12 ppm d Repeat a b and c for a 90 degree pulse length of 60 us on the same magnet e Repeat a b and c for a 90 degree pulse length of 60 pus on a 141 T 600 MHZ magnet Is the answer the same as d A S Edison Univasity ofFlonda 2006 Next Monday s Lecture 4 Mon Oct 9 Data Collection a Time vs Frequency b Hz vs PPM c Fourier Transform d Digitization and Spectral Width e Quadrature detection lson Unwasity ofFlonda 2006
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