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by: Guiseppe Bednar


Guiseppe Bednar

GPA 3.93

Robert Batey

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Robert Batey
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This 22 page Class Notes was uploaded by Guiseppe Bednar on Friday October 30, 2015. The Class Notes belongs to CHEM 4711 at University of Colorado at Boulder taught by Robert Batey in Fall. Since its upload, it has received 16 views. For similar materials see /class/232179/chem-4711-university-of-colorado-at-boulder in Chemistry at University of Colorado at Boulder.




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Date Created: 10/30/15
Lecture 8 3D structures Monday March 05 2012 1041 AM Three Dimensional Structures of Proteins A pulyrum 7 39 the 39 it s backbone Asa consequence of resonance interactions the peptide bond has a 40 double bond character 0 95 039 II c r c c C 4 S n r 4 ca T a C VN c T a H H H Either Cis orTrans Trans dominated due to sterics Torsion Angles Amid man sm gmuu r Amide Elma Wm M Lnryhaan4xvum a w l H mm CrN bond ct CrC bonerlJ 0 Some anglesare favored due to stability 0 Staggered mostfavorable Ll180 eclipsed least stable LlJzo 0 Energy barrier between the two is 12 mol 0 Substituents otherthan Hydrogen have greater energy barriers BioChemieryPage 1 Stericallyforbidden conformations o onrbonding distancelt van der Waal s distance Statistically observed in a Ramachandran plot In proteins only a subsat of phi and psi angles are observed which can be plotted on a RAMACHANDRAN PLOT 0 green regionsare lound In proteins are l l l secondary structure look like l l l l gt180 gt160 90 Helical Structures Rightehanded or leftrhanded point thumb in direction curl fingers around Alpha Heli Has allowed conformations and favorable Hebonding BioChemieryPage 2 4257 LJ 747 n36 residues perturn The backbone contains a DIPOLE moment Carboxyl terminus is 5 Amino terminus is 5 Beta Structures In B pleated sheetsl the hydrogen bonding occurs between neighboring polypeptide chains rather than Within one a he ix The second commonly found element of secondary structure found in proteins is called the B sheet which involves J hydrogen the polypeptide backbone E L I I L I I A 20 b L A 6 conformation called the betastrand 46 0 K a 0 si e o e w 9 a a 0 a a r 0 n 3 o r I hr 0 the vstis lp39ithesecond39down39ithethird39hp etc Two Varieties o Antiparallel pleated sheet inwhich one neighboring ydrogen bonded polypeptide chains run in opposite directions 0 Parallel 3 pleated sheet in which the hydrogen bonded chains extend in the same direction a Anhparallel r V 7 Top view ax i5 Side View BioChemistryPage 3 I Paraiiei B sheet of iessthan five strands are rare suggeststhat paraHei is iess stabie than antiparaHei possibiy bc Hrbonds are distorted in comparison I Requires ioops to go to next B sheet as shown beiow as 52 oflha repetitive slruzlure of the sheet them m 5 be LOOPS helween that autumn the Individual strands whlch further dictate the mpniugy of the protein 5 Di 2 a HAIRNN unnemon b a ngm handed crossover It 3 led handed lassaver In manypmtelns the q vars In C adopt I ha helkal mnlurmaunn Can aiso use a Bturn Biochemistry Page 4 The tight turn of betahairpins adopts one of seve I conformations to create a stable structure called betaturns a 3 Turns Type II Nonrepetitive Structures yulu 39 L 39 2 H39 half of the average globular protein Coil or loops 0 Less repetitiveordersthan a helices 0 Random coil totally disordered and rapidly fluctuating set of conformations assumed by denatured protein Reverse Turns and B bends Often connect successive strands of antiparallel B sheets Fibrous Proteins L Hui structures are their dominant structural motifs ratinA Helix 0d Helices o Mechanicallydurable 0 Ex Hair horns nails etc 0 Rich in Cys residues forms disulfide bonds that cross link Due t Cys also insoluble and resistant to stretching very importantfor biology Hard or soft due to amount of S content I Hard more disulfide bonds that resist any forces trying to deform them Biochemistry Page 5 Keratin a helix Two chain oiled coil Proto lament Protofibril The twohair coiled n 2 a K a a 2 t quot 450Ainlength These 1 mde hmem bquot Pm II 4 4mm 3 939 organization to create r lmnhain oiled tall gx J u Hullx Cross section of a hair 39 r sun 1 5 If 9 53 SH H reduce ml 5quotquot uxudlxe as was sm lI 5 5quot us H 5H us 3 M H 595 7 sm 4 a 965 SS SR 65 a Collagen o Occurs ln all multlcellularanlmals o Extracellular proteln that ls organlzed lnto lnsoluble bers of great tenslle strengt 0 Stress bearlng component of bones and teeth 0 Three malnty es 39 Type I a1l2alrslltln bonester1dor1 blood Vessels cornea 39 Type II a1ll37 car tllage lntervertebral dlsllt 39 Type III ul37 blood Vessels fetal slltlr1 0 Nearly 13 are Gly 0 15730 Pro and Hyp Arhydroxyprolyl o Hyp req ulres ascorblc acld Vltamln C Biochemistry Page 6 Further stability is conferred by hydroxyproline a posttranslational modi cation ofthe protein which requires ASCORBATE Vitamin C Pmlvl mum mKLuglulnule Pm gtnoysse V A cuum m nrnydwn wolyl residue HO H 0 CicHyon H i V HO OH HO L Ascorbic acid Ascorbate H0 H r 39Han NH l w u Deliydroascorbic acid Scurvy the collagen synthesized cannot form fibers properly Structure of Collagen Collagen39 5 39 39 39 39 39 L39 L 39 39F quot quotL quot MW 39 39 WlLI39I gentle rightrhanded roperlike twist toform a triple helicalstrutture Biochemistry Page 7 Three strands are wound together to form a triple helix O The staggered peptide groups are oriented such that the NH of each Gly makes a strong Hibond with the carbonyl oxygen of an X pro residue on a neighboring chain Packed into a fibril structure Drivingforce forthe assembly is the added hydrophobic interactions within the fibrils in a manner analogous to the packing of secondary structure to form a globular protein Cunang umlemlle gumm i Packmg of molecules 00 memmec A L OQQQK smmm 273920 LTNXW 1mm smmw i9 0 Covalently crosslinked O Derived from Lys and His residues not Cys like in Keratin Screwed up collagen causes defects 0 Mutations of type I collagen causes brittle bone disease 0 Screwed upfibril structures are implicated in Alzheimer s Can also be in B sheetform BioChemieryPage 8 Fibrils an also be formed from beta sheet structures K broin i 393 4P g J 35 A1 33 9 5 57 Almwquot 0 3 39 w 0 Jig lu 5 1JW W39 5393 tau a a h 0 N3 Ala side chain Gly side chain quot ummln nranuwhle envy um n I v 4 BioChemieryPage 9 Lecture 9 Proteins Monday March 05 2012 1209 PM Globular Protein Highly diverse group ofsubstances that in their native states exist as compact spheroidalmoiecules The tertiary structure of a protein describes how the various elements of secondary structure are packed together to create the overall topology of a protein in its NATIVE state Various representations of myoglobin Interpretation of Protein XRay and NMR Structures Xrays Xray crystallography First you must generate ordered CRVSTALS at the protein of interest AdJmmI ui R H mm i EstMai dew gt s a an i o i x x V 4 9 4 Myoglnbin in Myuglobin in Myoglobin crystals dilute bullet 3 M NHQJSDA pH 7 unruly In an best thought of as a slow orderly precipitation process Biochemistry Page 1 00000000 This generates a DIFFRACTION PATTERN each re ection aka spot provides information about the shape of the electron cloud surrounding the molecule of interest me Xrays interact aimost exciusiveiy with the eiectrons in matter not nuciei Image ofeiectron density iiilte atopographic map a toy uiume This gives them a soft jeiiy iiilte consistency Resoiution iimit 0f153 Angstroms it i easier quot quot 39 39 39 39 iieauu Hydrogen s are usuaiiy not seen since they oniy have one eiectron BiochemistryPage 2 o A protein molecule in a crystal is essentially in solution bc it is bathed in solvent 0 Crystal packing forces do not greatly perturb the structures of protein molecules NMR gives same sha e 0 Many enzymes are chemically active in solution R 0 2D Nmr o Consistent with an ensemble of closely related structures From these data a family of models consistent with all of the experimental information is derived Note the fuzzyquot regionsoften regions of conformational flexibility Vi myoglobin 0 NMR methods are limited to determining the structures of macromolecules with MW of no greaterthan 40 kD some day maybe bigger 0 Surface residues may cause disparities between nmr and xray since they participate in intermolecularcontacts and are thereby perturbed from their solution conformations 0 NMR can be used to probe motion overtime scales and be used to study protein folding and dynamics Tertiary Structure Other proteins can contain betasheets or a combination ofalphahelical and betasheet structures concanavalin A carbonic anhydrase May contain both a Helices or B sheets Most proteins have a significant portion of B sheets 28 and a helicies 31 Side chain location varies with polarity O Nonpolar residues Val eu ile met phe largelyoccur in the interior ofthe protein out of BioChemieryPage 3 contact with the aqueous solvent bsthey re hydrophobic Duh 0 Charged polar residues Arg His Lys Asp Glu are located on the surface incontact with solvent bc the immersion of an ion in the virtually anhydrous center of a protein results in the uncompensated loss of much of its hydration energy D The uncharged polar groups Ser Thr Asn Gln Tyr and Trp are usually on the surface but frequently occur in the interior In the latter case these residues are almost always hydrogen bonded to other groups This is a mess But there are some underlying organizing principals to understanding how tertiary struttuve is established LOCATlDN OFTHE SlDE CHAINS HYDROPHOEIC residues Val Leu lle Mel and Phe are generallyfound in the interiorofa proteinwhile CH amino acids are found on the exteri while me ilgni shows a IDSSVSECIIDH quot3100917 the interim oiLhe pimeln Globular Protein Cores are Effi 39ently Arranged with Their Side Chains in Relaxed Conformations o The interiorof a protein is CLOSE PACKED to exclude almost all water and dis o 5 formation of internal cavities optimized van der Waals packing of all atoms The packing density of a protein is 075 versus 074 for closerpacked spheres Large Peptides Form Domains quot 39 39 quot391me L L globularprotein SupersecondaryStructures are the Building Blocks of Proteins otif a small Most common I Rightrhanded crossover connection between two consecutive parallel strands of a B sheet and consists of a a he 39gtlt o B hairpin motif I Antiparallel B sheet formed by sequential segments of polypeptide chain that are connected by relatively tight reverse turns 0 immoti 0 Two consecutive antiparallel a helices pack against each other with their agtltes inclined so as to permit their contacting side chains to interdigitate efficiently Biochemistry Page 4 that are mmmonly found in many pmuins samnlimns rafeued w as SUPERSECONDARV STRUCTURES 39j t r 39 lt1 I D u Loop I 1l Corner 5435 loop 5 mm These smaller units can be easily found in more complicated structures 3 3 Loop u Barrel 1 Domains 0 Globin foldisahelices o 4rheigtlt bundle B domains 0 Bsandwich 0 Up and down barrel al Barrel O B barrel repeated use of beta hairpins BioChemieryPage 5 n Qualernz ry structure QUATERNARV STRUCTURE m Purl nl mmm armrer mmmm pappepuarmvm menu mm a Imam mm Msmmvmnrmu quotpk m yarpm Pvmmv 39rhm Wm Wm Adm1mm mm uvvul uLmIakumumw mm A mwammarene mm o PrDtEms WKH dEnUca subumtsmhgumers pmtnmErthEszdEntma subumts x m Er A2132 0 Symmetry MammyBfDthmEncpmtEmsthEpmtumErsarEsymmancaHyarra ged FrDtEmscan nn yhavermatmna symmarybcsuchsymmetrynperatmnscnnven chm erEswduEs m drrEswduEs o Cychcsymmztry More omplex quaternary swunures are th result of many dividual polypeptides packing into a symmem array Twofold Threefvld Two types of cyclk symmetry 0 D hEdra symmary EmchzmxskyPAge Twofold Fourfold Twofold 39 Kadlwofold Twofold 4 Two types of dihedral symmetry 0 Helical symmetry BioChemieryPage 7 Lecture 7 Structure of Protein Thursday Januarv26 2012 631 PM 4 levels of organization Primary Stmcture Determination of Proteins 0 Primary amino acid sequence Helpful because 0 Secondary local spatial arrangement ofa polypeptide39s 0 Knowledge ofa proteins amino acid sequence is essential for atoms without regard to conformation Ex Alpha helix an understanding of it39s molecular mechanism of action and 0 Tertiary 3D structure is a prereq for NMR and Xray crystallography 0 Quaternary spatial arrangement ofmultiple 0 Can yield important insights into how proteins function and polypeptides how they evolved 0 Many diseases are linked to mutations in amino acid The structure ofproteins an be described on four levels sequence Determination Technique Steps 1 Prepare amino for sequencing a Determine 1 ofchemically different subunits in protein b Cleave peptide39s disulfide bonds c Separate and purify unique subunits d Determine the subunit39s amino acid compositions 2 Sequence polypeptide chains a Fragment individual subunits at specific points b Separate and purify c Determine amino acid sequence for each fragment d Repeat ZaZc with different fragmentation points 3 Organize completed structure a Span the cleavage points between one set of peptide fragments to the other b Elucidate positions ofdisulfide bonds End Group Analysis Different types of Subunits Each polypeptide has an N terminus and a Cterminus BY identifying each we can determine the number ofdistinct polypeptides in a protein N terminus Identification O Dansyl Chloride I Reacts with primary aminesincl amino group on Lys to yield dansyl peptides Acid hydrolysis liberates the nterminal residue as dansylamino acid Intense yellow 0 Edman Degradation I PITC reacts with nterminal amino groups of proteins under mild alkaline conditions to form PTC adduct This is treated with an anhydrous strong acid trifluoroacetic aci Which cleaves the nterminal residue as thiazolinone It is selectively extracted into an organic solvent and converted to PTH derivative The amino acid chain is identifies by comparing its retention time on HPLCwith known PTHamino acids I The Edman degradation therefore releases the Nterminal amino acid residue but leaves the rest of the polypeptide chain intact he chemistry of the reaction I Pros D It is able to accurately sequence up to 30 amino acids with modern machines capable of over 99 efficiency per amino acid D Only uses 10 100 picomoles of peptide for the sequencing process D Automated to speed up the process BioChemisLIy Page 1 D Removes sequentially the Edman degradation thus removes amino arids sequentially M rm mlImmldnlu hnrnnuRl m m umm mm mm w WWW n m Hutu um imam I Cons D It will not work ifthe Nterminal amino acid has been chemically modified or if it is concealed within the body ofthe protein B Guesswork ora separate procedure to determine the positions ofdisulfide bridge D Concentrations of 1 picomole of peptide or above for discernible results C Terminus Identification o Enzymes I No reliable way must use enzymes using exopeptidases enzymes that cleave terminal end Carboxypeptidases catalyze the hydrolysis of the Cterminal residues Pros highly selective I Cons cannot be used to determine sequence because they also cleave side chains Enzymes i mm 414 V m winlun um l mu yr mu 0 Chemical Methods I Treated with hydrazine at 90C in the presence ofa mildly acid ion exchange resin I Lots of side reactions Cleavage of Disulfide Bonds Done for 2 reasons i To permit the separation of polypeptide chains ii To prevent native protein conformation from obstructing the action of protein cleaving agents due to stabilization from disulfide bonds Cleaved reductively by Zmercaptoethanol Dithiothreitol or dithioerythitol Cleland39s reagent Free sulfhydryl groups are then alkylated with iodoacetic acid to prevent reformation of disulfide bonds OOO 5 M I l quot31gt Diaulldelmlmddmiu 1 IndianInt thln irmnl i BioChemisLIy Page 2 l 53 le gtA1LgtquotJilulns Dlnu ldu Inlle Lil1H1 Iodumun ulxhimmnul m m 9mm ml Inn39mnhymwl mm Amino Acid Composition The amino acid composition ofa subunit is determined by its complete hydrolysis followed by the quantitative analysis ofthe liberated amino acids 0 Can be accomplished by acid or base or enzymatically Acid Catalyzed Hydrolysis Polypeptide is dissolved in GM HCL sealed in an evacuated tube heated to 100Cfor 10100hr Cons 0 Ser Thr Tyr are only partially degraded Destroys Trp residues Gln and Asn are converted to Glu and Asp plus NH4 OnlyAsx AsnAsp and Glx Gln Glu NH4 AsnGln are known Base Catalyzed Hydrolysis 0 Polypeptide is carried out in Z ro 4M NaOH at 100Cfor4 8hrs 0 Principally used to determine Trp content 0 ConS39 O o Decomposes Cyc Ser Thr Arg 0 Partially racemizes and deaminates other amino acids Enzymes 0 Endopeptidases catalyzes hydrolysis 0 Commonly Pronase 0 Limited to 1wt because enzymes will selfdegrade 0 Used forTrpAsn Gln System has been optimized with Mass Spec see below Amino acid compositions of proteins are indicative of their structures 0 Leu Ala Gly Ser Val and Glu are the most common amino acid residues 0 His Met Cyc and Trp occur less frequently weird ones Specific Peptide Cleavage Reactions 0 Trypsin 0 Greatest specificity o Cside of the positively charged residues Arg and Lys ifthe residue is not followed by Pro 0 charge on Lys is eliminated by citraconic anhydride and Trypsin does not cleave 0 Can be deblocked by mild acid hydrolysis O Cys can be made to have a charge is aminoalkylated by a betahaloamine 0 Cyanogen Bromide Cside of Met 0 Forms petidyl homoserine lactone O 1 M HCl that denatures proteins so that cleavage normally occurs at all Met residues Mass Spec 3 w Malditof 1 Protein sample is ionized dried Z Irradiated with laser 3 Electrical field accelerates ions 4 5 LIghtest ions arrive at the detector first Laser triggers a clock Biochemisuy Page 3 gt300 kD have been characterized MALDITOF mass spectrometry This yields a mass spectrum of the protein of interest which is identi ed based upon its mass because of the very high accuracy of the measurement error1 part in I mxnir ii ll 1 id mumquot ii AW MEIR L mni m u ESI electrospray ionization Protein sample ionized at high voltage 2 Fed to a high vacuum with inert gas that the solvent evaporates 3 Mass analyzer does a mass per charge r u 39 39 W7quot r5 W mm mgr Mm Biochemistry Page 4 The mass spectrum of a protein looks quite different by ES clue to the greater degree of ionization of the protein SCCI HG each peak differs from the next by a charge C GU g dlfference of one and a E mass difference of one g m Lieu a proton ix 20 wt tril l l 39 x z r 7 it 503 330 1000 170 lle 16 lRl A l 7 0i mi Tandem 1 Protein sample ionized Z Onlything you want hits collision cell MSl 3 M52 detects output quotui Electrostxay ionization tandem mass spectrometer quotEmu m J Elsclrnspxay NS le sirm Cell M2 Detector leZallDil SJLII39CE Coillsrou ccxl n It or several of its peptide bonds 39m mxi mu Biochemistry Page 5 Peptide Sequencing By Mass Spec Short peptides lt25 residues can be directly sequenced Hard to do others because inherent error compounds with each residue Using this method we can identify individual proteins from complex mixtures of proteins that have been separated by ZDelectrophoresis silver stained an individual spotfrom the gel excised and analyzed by mass spectrometry Biochemistry Page 6


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