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Midterm 1 Study Guide

by: Shreya Sreekantaswamy

Midterm 1 Study Guide 14263720

Shreya Sreekantaswamy

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Here is a detailed Study Guide that covers all the topics tested on for Midterm 1. Key points are bolded or in red, vocab in light blue, and examples in purple. Graphics are concluded.
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This 17 page Study Guide was uploaded by Shreya Sreekantaswamy on Sunday November 2, 2014. The Study Guide belongs to 14263720 at University of California - Los Angeles taught by MORIN-LEISK in Fall2014. Since its upload, it has received 202 views. For similar materials see BIOCHEM 153A in Biochemistry at University of California - Los Angeles.


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Date Created: 11/02/14
153A MIDTERM 1 BASIC REVIEW 0 Carbon can make 4 bonds o 4 single bonds tetrahedral o 1 double bond 2 single bonds trigonal planar I No free rotation about a double bond 0 IMPORTANT FUNCTIONAL GROUPS H Emma H 39 H limisslamle Fl2 fl FlI39lil f39I 1E39E H HHHVVEH ill I H H N Er Amizln H p lil I U Di5lJiii 25E 0 Biochemistry branch of science concerned with the chemical and physiochemical processes that occur within living organisms o Includes components of I Cell Biology structural feature common to all cells I Genetics sequence defines structure central dogma I Physics free energy kinetics I Chemistry carbon and functional groups biological macromolecules I Evolution first biomolecules RNA world mutation and natural selection 0 3 main domains of life Bacteria Prokaryotes Archaea often extremophiles Eukarya eukaryotes membrane Features Common in all Cells Bacterial cell Animal Cell Cytoplasm NO MEMBRANE BOUND Many membrane bound Plasma Membrane ORGANELLES organelles Ribosomes Nucleoid genetic material Nucleus with nuclear membrane confined to middle of cell but no Golgi Apparatus ER Lysosomes o Prokrayotes eukaryotes both have PACKED cytoplasm 0 Free Energy AG amount of energy available to do work 0 AG Gproducts 39 Greactants o Gibbs Free Energy AG AH T AS I AG EXERGONIC spontaneous releases energy favorable I AG ENDERGONIC requires energy unfavorable I AS more disorder I AH heat released AS less disorder AH heat absorbed 0 Equilibrium reached when rate of product formation rate of reactant formation a o aAbBe cCdD CquotDquot e T ra IOO concen ra On reac e W en equll rlum IS reac e Ina K q AaBb t f t t h d h lb h d REVERSIBLE RXN o Steady State aA lt 5 bB I All state variables are constant requires steady flow through system AG overall free energy change AG overall free energy change for physical chemistry AG overall free energy change in biochemistry EQUILIBIRUM AG o E R K 39 REVERSE FAVORED AG gt o E gt Keq R FORWARD FAVORED AG lt o E lt Keq R GENETIC amp EVOLUTIONARY FOUNDATIONS 0 Linear polymer of DNA contains instructions for making every cellular component 0 Structure of a molecule help define its function 0 THE CENTRAL DOGMA llIex ki nisase gene DNA 0M V pc Y I I 1 z rain EE ri39pti39uin of in IMA T initsci Eumpllementary EMA RNA Can edit self quotlII Ei1I391 lluT ItiiiilI i of RNA can 1 1ribua5uime to pnllypeptide chain A A PROTEIN ATP giluEn SE ADP gliulcuse Eatall rtiEalI r g Flh iphafe active hemkinlasse 0 Miller amp Urey Experiment generate first biomolecules abiotically from a primordial soup then evolve chemically 0 Mistakes are made during replication selective pressure allows the organism to evolve the new gene to adapt to changes in its environment WATER 0 70 of body mass planet mass 0 Forms Hydrogen bonds weak individually but strong combined with solutesmacromolecules itself A 0 sp3 orbitals roughly tetrahedral 1045 similar to a methyl carbon 0 electronegative Oxygen allows for partial charge formation o makes water molecule polar but overall uncharged 0 product in condensation reactions usually endergonic ex ATP synthesis 0 reactant in hydrolysis reactions uses H20 to break bond exergonic ex RNA cleavage POLARITY 0 bond is polar if there is a large difference in electronegativities of bonding atoms 0 needs dipole moment asymmetrical distribution of charges WEAK INTERACTION 1 HYDROGEN BOND 0 alone are weak but summed together give liquid H20 substantial cohesive properties 0 water can form 34 H bonds with other water molecules H i i T o contributes to high melting and boiling points is H d quot39Equotnl quot d 139n A 23 litllr irImo39l 0 length of water Hbond measured between 2 EN centers includes the covalent bond 3 3 of 0 and H l maialeinit lmmtll RULES I ggquotf39i il gTmm o Requires EN donor N O S that has covalent H E 0n 3 Requires EN acceptor N O S that can be fullypartially charged Relative to covalent bonds easily broken and reformed O H o Ideal Hbond length 27 34 A between EN centers ti V II o The more bonds the more stable I l5tquot 3939lquot9 H Wealker o Angle Important for bond strength 39 hydlmgien EN Mdmgen quotequot393 l RD c 3 band i hann 7 I WEAK INTERACTION 2 IONIC INTERACTIONS 0 Attraction between oppositely charged ions or molecules ex NaC 0 Hydrophilic chargedpolar compounds that dissolve easily in water 0 Water dissolves salts by replacing solutesolute ionic bonds with watersolute polar interactions o Energetically favorable because increases entropy split cation and anion WEAK INTERACTION 3 HYDROPHOBIC EFFECT 0 Hydrophobic non poar molecules with no net dipole moment do NOT dissolve in polar solvents 0 H20 forms highly ordered cage around nonpolar molecule which decreases entropy o When there are multiple hydrophobic molecules they clump together said to interact even though NOT forming any bonds water molecules form one BIG cage around the cluster I Increases entropy because less molecules are needed than if were to make small cages around each separate hydrophobic molecules 0 Amphipathic molecule that contains both polar and non polar functional groups o Hydrophobic faces interior hydrophilic faces aqueous environment WEAK INTERACTION 4 VAN DER WAALS INTERACTIONS 0 Random variation in electron clouds of 2 atoms can create transient dipole leading to weak attractive force bringing 2 nuclei closer o Said to experience van der waals interactions at point where attractive force is maximal o As overlap electron clouds experience strong repelling force min distance before repel van der Waals radius 0 Van der Waals induced by London dispersion forces debye force induced dipoles kesom force permanent dipoles WEAK INTERACTION SUMMARY 0 All individually weak compared to covalent bonds 0 Cumulatively very strong 0 Dynamic constantly breaking and re forming 0 Large contribution to macromolecular structure IONIZATION 0 Important in macromolecular structures proteins lipids nucleic acids all depend on acidic ionization of functional groups ex acidic phosphate in DNA backbone ionizes so negatively charged at pH 7 negative charge impacts helical structure 0 Important in many biochemical reactions several reactions require ionization of waterfunctional group to catalyze reaction ex serine proteases loses proton giving formal negative charge required for reaction to proceed 0 Water capable of weakly and reversibly ionizing to generate H and OH ions HZOH H OH o Free protons in a solution are ALWAYS hydrated by another water molecule H20 H20 H H30 OH o Commonly write HA gt A H 0 Reversible reactions are essential to cellular function f quot 0 Kw ion product constant of H20 10 1039 M HOH 1 G39sIriiiicE 2 A Lemonjuice 3 y quotI iw e Cola vinegar pH 4 Iquot39cf gIy g ed wine V Beer o ogH ST gtBilackzrffee T T n p 7T Nequotquotquota 39 0 y P z tears 0 scale is logarithmic ie pH decrease of 1 means 10 fold increase in H in solution 3 Seawatereaisiwhite 9 5oIution ofibakiixng soda ihlallcclgl ACIDS 0 acid with greater tendency to release protons considered stronger acid Household ammonia 0 Ka acid dissociation constant equilibrium constant for ionizing acid quot5e i 39 i 39Ea H A 1 M NaOH o HA H A Keel HA Ka o HAH A Keq 3 pKa 39 gKa o the stronger the tendency to dissociate a H the stronger the acid the lower the pKa 0 Henderson Hassebach pH pKa log E o In some cases protonated molecule is charged form HA while unprotonated is neutral A AMINO ACIDS AA building blocks of proteins coo Carboxyl group H IE CI H Alpha carbon carbon to which Ammo 3r F N 3 functional groups covalently I attached R side chain R unique for each AA 0 main chain quotbackbone atoms N alpha C CO 0 all AA except glycine are chiral o non superimposabe mirror images enantiomers 0 identical physical properties except rotation of polarized light 0 very different biological properties 0 quotL form NH3 on left side is major form of AA in biological systems 0 Hydropathy Index whether or not free energy change is favorable when you move from hydrophobic to hydrophilic environment o value hydrophilic value hydrophobic El 0 Important modifications that can affect proteins activity 1 an E o Phosphorylation reversible in STY very important for cell signaling can regulate protein activity o In plant cell walls H U I Ho rf2H IgEEfCH E 39 39 39 H Hl IIH3rdr39mqriprulliinie n co agen llI 3M cH tn Hl CH3 EHg tilH coo DH tum 5Hycl rmqrl3r5i ne In myosin WH N Meitlhyllysine 3 H 0 Carboxyl amp amino groups are I l l 0 Iomzable AA can donate or H C CODE accept protons met llIlH3 0 Zwitterionic at pH 7 have both ghargi g 1 amp charge o Common formal charge q O o Nonionic form is rarely observed c our C on coo I nonpolar R groups l I l K PL c I J H CH I rll C EH CH HlgllI C H H3Ill C H y39c Ill3II C H I 2 l 3 I 1 l l H EN CH2 l on CH EH1 H CH3 l CIIi CFI CH 1 l H 2 cH2 cI3 cH3 so 3 3 CH3 Glycine Alanine Proline Valine CH3 jT 11 F V Leiucine lsoleucine Methionine L I HI 0 G small achiral very flexible 0 A V L I large hydrophobic effect often buried beneath protein surface away from solvent 0 P cyclic side chain with covalent bond to amino group NOT FLEXIBLE 0 M sufur containing thioester non poar o Because is followed by a CH3 group not as EN to Hbond aromatic R groups 0 All have substantial hydrophobic effect and absorb UV light 0 F nonpolar phenyl side chain 0 Y W Hbond 0 W iodole side chain 0 Y phenolic side chain B equot39amber 39aquotquot Ae 39 Plhenylllalanimel Tyrosine Tryptophan IF Y W o A absorbance e extinction coefficient M391cm391 c concentration M I light path length cm o Allows to measure concentration of a protein o E is additive I Ex for 3 Trp 2 Tyr Betrp zetyr polar uncharged R groups C GD C DD EEID 0 II P H3lll H H3lquotlH H3l ilH CHEGH HEEEDH H2 CH3 SH Seriine Th reoiriine Cyslteilnie o T I 0 Soluble in water and can form H bonds 0 S T hydroxyl group 0 N Q amide groups no charge 0 C S H is a sulfhydryl group weak acid ionizes to S coo coo l IlI3N ZHI llI3NCIlll C CH V 1 HEN o C pS Hglll o Aspairagiine Gllutaimine NI 0 o Two neighboring sulfhydryl groups can oxidize to form a covalent disulfide bond apolar hydrophobic usually stabilizing i F i negatively amp positively charged R 00 00 3quot39 EH 3quot EHquot 3quot EH group lll3lN fT39 H H3NH quotlNII 0 Polar water soluble and ionizable EH2 H 2 1iH2 IZH2 p CllLl carboxylic acid R group 09 CH 3 ml 0 Net charge 1 at pH 7 gg 1In3 iI 0 Side chains form stabilizing ionic ggpamate Glutamate H2 interactions with charged side D E Ly e Argglme Histildine chains c out coo coo I I l H3NyC H Him c H iH3hl C H 0 Polar water soluble and ionizable amine groups cu EH2 EH2 0 K R have significant hydrophobic component EH2 EH1 NH 0 H very important since it can be charged at pH 7 and facilitates Elm EH2 cH many enzymatic reactions C H IEEN 0 Side chains form stabilizing ionic interactions with neg charged side lly liz chains S lm Lysine Arginine lllistidine K R H TITRATION CURVE 0 Acidity constant Ka must be measured by titration of pH 39iWH Elm biting Halal IH H 3N1 Cli Hal tH HHvEiH o Strong base NaOH is added to known volume of weak h H EH2 H ill H H acid 2395 inn m 3 pit ig pie Ellljhlg gi o As base added removes proton from the acid and f E53 5 l 13 5 9 increases pH 2 11 0 1 0 KEY FEATURES W H551twine PK 39 o Equivalents xaxis given in mol base mol acid Agm 1 I O1 monoprotic O2 diprotic O3 triprotic a E PK E l 9 as y y y y 65 E E o Equivalence Point complete deprotonation has PH 539 quotf5quot 1 occurred 4 If o lnflection Point pH pKa for acid A pllfj 5 RULES 2 39PE 5 o Within 1 pH unit is good buffering region 1 w w y E o If pH measuring at is lt 2 pH units is fully protonated 0 Ln 2 li 3li o If pH measuring at is gt 2 pH units then fully de DH 39EEllUi39H39EIElM5l protonated pl ISOELECTRIC POINT 0 pH at which net charge 0 o to calculate find 2 groups that straddle net charge of 0 and average their pKa s BUFFERS 0 aqueous system that resists change in pH when acidbase added o critical for maintain pH of cellular systems 0 buffer works best at 1 pH unit flat part of titration curve 0 maximum buffering capacity at pKa 0 most cells use small molecules or proteins to act as buffers and maintain pH at constant level between 69 and 74 Peptide bond PEPTIDES 0 Chains of AA linked through condensation reactions between carboxyl acid and amino groups 31 o Makes peptide bond amide n in 0 Molecular weight MW of a peptide MW sum of AA minus MW of water 18 Daltons o E1MW n 118 o Polypeptide with MW gt 10000 Da is considered a protein an amide 0 Peptide bond results from condensation reaction between COO and NH3 of 2 amino acids o C amp N spz hybridized planar o Partial neg on O and partial on N makes it slightly polar no free rotation about peptide bond o Bond conformations cis alpha carbons on same side of bond trans alpha carbons on OPPOSITE sides of bond 0 Because of steric hindrance trans form predominates quot39 10001 phi 25 I o EXCEPTION PROLINE TRANS FAVORED ONLY 51 0 Phibond CNCaC psibondNCaCN o Unlike peptide bond phi amp psi bonds have free rotation 4 psi bond w o Phi amp psi angles torsion angles o Are inhibited by steric interference at certain magnitude SEQUENCE OF AA 1 STRUCTURE 0 Sequence of AA defines proteins 3D shape shape gives functional properties 0 Edman Degradation method for sequencing proteins 0 STEPS I Label Nterminus with PITC I Cleave off Nterminal residue and separate it from remaining peptide I Identify the AA by HPLC high pressure liquid chromatography I Repeat reaction with remaining peptide to determine the new Nterminal residue o Efficiency limits reading to 5 AA 0 Digest protein of interest with various proteolytic enzymescompounds o Use DTT or betamercaptoethanol to break disulfide bonds o Trypsin cleaves after K R o Cymotrypsin cleaves after F Y W o Cyanogen Bromide cleaves after M o V8 Protease cleaves after D E 0 When protein sequence is similar across many species said to be conserved o Consensus sequence most common residue at a specific position upon alignment of protein sequence from several different species 0 Signature Sequences sequences that are unique to specific proteins that are mostly conserved RAMACHANDRAN PLOT Theeuretital panl i39 al3 0 Plot of Q5 and Lu angles showing sterically aowabe conformations Hm a 0 Kind of like a heat map showing where the angles are common S PROTEIN 2 STRUCTURE quot339 0 Stable reoccurring arrangement with regular Q5 and w angles for residues in the 5 element 11 quot 0 Protein folding El T U 1 E 0 Adopt lowest energy conformation iildle feffl o Maximize main chain Hydrogen bonding amp van der waals Amino terminus o Minimize steric clash alpha helix 2 0 Has a dipole moment all Hbonds have polarity in the same direction o C terminal CO does not make any bonds so has charge 1 o N terminus NH does not make Hbonds so has charge 1 0 Note that all other CO and NH participate in Hydrogen bonding 0 Charges are additive the longer the helix the greater the dipole moment 0 R handed helix 0 Atoms in helical core are tightly packed maximize van der waals 0 Side chains are pointed out away from helical axis Carbonyl terirnin us 6 la 0 HELIX STABILITY o Some AA adopt D and Lu angles that are favorable for the formation of alpha helices I Ala good Pro bad Residues with the same charge repel and disrupt helix Residues with opposite charges attract and stabilize helix I Separate charges by at least 4 AA Amphipathic Helix helices that are hydrophobic on one side and hydrophilic on the other Helical dipole partial positive amp partial neg charge stabilized by having opposite charged side chains at that end I ie at Cterm Arginine at N term Aspartate beta sheet 2 0 Beta Conformation extended zigzag conformation 0 Beta strands stack side by side to form beta sheets o Strands connected by Hbonds o Side chains alternate above and below quot ti Pal39aquot39939 5 PA 1 Paramquot plane I C 4I 0 Beta turns region of protein involving 4 consecutive residues where polypeptide chain folds back on itself 180 degrees o Type 1 contains proline o Type 2 contains glycine o If residues defined by positions i i1 i2 i3 turn involves Hbonding between i amp i3 residues RANDOM COIL 0 Region that does NOT adopt 2 structure though not really random 0 Reduces overall free energy CIRCULAR DICHROISM differential absorption of left 25 39 39 39 and right polarized light 29 39 0 V 0 Allows spectroscopic determination of 2 structure conformation 15 0 Scan of wavelength in far UV region where peptide bond absorbs Iii plotted against Ae difference in molar extinction coefficients of L 3 S B and R handed plane polarized light D i A tanlfarmatium andom coil o Different 2 structural elements give a signature curve o Used to assess folding of denatured protein 30 4 quotl939El Z ll 210 220 230 240 25D 0 O wavelength nm 0 3 composed of 2 structural elements connected by random COIIS o Consists of weak interactions largely hydrophobic effect 0 Motif supersecondary structure different folding patterns that consist of one or more 0 4eh ll bundllle DHSGlll d mil 2 structural elements y I y o Stabilized by Hbonds van der waals hydrophobic effect 0 Can be amphipathic ieme x Incup BarrIearIder 0 Large motifs can be constructed from smaller ones ie all alpha all beta alphabeta 0 Domain distinct functional unit of protein that can exist on its own 0 1 motif or more can make up a domain 0 STABILIZING FACTORS o Hydrophobic residues in protein core water doesn39t have to make cages around hydrophobic molecules entropy is increased so energetically favorable 0 Maximum van der waals contacts in core gaps in core will be filled in due to attractive force between atoms Hydrogen bonded 2 structures maximized Hbonding and van der waals contacts in main chain atoms Amphipathic 2 structures increase hydrophobic interactions in interior increase Hbonding with water or other residues on the exterior 0 Reverse turns beta turns allow for peptide chain to reverse direction requires unusual conformational flexibility provided by prolineglycine and Hbonding o Disulfide bonds covalently crosslink 2 Cys residues that may be very far apart in 1 structure but close when folded in 3 0 Long range Hbonds similar effect as a disulfide non covaent QUARTERNARY STRUCTURE 0 Consists of 2 or more separate polypeptide chains associating with each other in a specific manner required for optimal activity Dimer trimer tetramer hexamer 0 HomodImer 2 polypeptides are the same dodecamer 0 Heterodimer 2 polypeptides are different PROTEIN FOLDING 0 Proof for sequence determines structure when you completely unfold a pure protein it can be re foded without additions of other factors 0 Denatured protein unfold a protein through heat pH changes organic solvents o Inactive state 0 Native protein folded and active protein 0 FOLDING MODELS 0 Some proteins use chaperones usually requires ATP 0 Stepwise Model I Local 2 structure I Hydrophobic effect causes packing I Longrange interactions motifs then domains 0 Hydrophobic Collapse Model I Drastic collapse into compact but dynamic form Molten Globule I Largely dependent on hydrophobic effect I Some 2 structures but mostly side chains NOT in final form 0 Misfolding can occur but then is usually degraded when it isn39t then can lead to diseases o Alzheimer39s extracellular deposition of amyloidosis Beta sheet plaque of amyloid fibers o Prions mis foded form catalyzes misfolding of other prion proteins 0 Folding is energetically favorable gt reason that proteins are stable o When go from unfolded state to folded have decrease in free energy METHODS OF PROTEIN PURIFICATION subcellular fractionation 0 Release proteins from cells through cell lysis 0 Go through differential centrifugation successive spins in centrifuge at higher and higher forces to separate pellet and supernatant E i v BiiI E39 239 21 I r quot1nunism quot15niann a 1llmin P 15lrrIiri rriirl 39 Elmr 1aquot1 397 Eupuarnatmrnri I W y y F It L A FaIlatrial1ilquot i pK 3 iiquot 39 Feliatriizihilin ni rneumesquot F lll tlfl an a A 0u N itrmhnndrla Ipltle mam l39e7 LllTIlE EllHI lilI o c Tiaaue Harri I name I GEIIHIM glam Egllihgggnplsml EirIm a39nea nriI c 39 quot fr quotlantV uaalllwntennsal EEME V gimpquot 1 meli1m4a column chromatography 0 Run proteins over a matrix and different proteins bind to the matrix differently 0 Can use either FPLC Fast Phase Liquid Form HPLC High Performance Liquid Chromatography 0 HPLC tends to use higher pressures 0 IonExchange Chromatography charge o Cation Exchanger column matrix anionic negative charge I IlLfarg2 net posiwe charge G Net punitive charge E llillert netgallgiiiim ltjliijairgue o Anion Exchanger column matrix cationic positive l lllargE net negative ull rargE charge b o Proteins interact with matrix according to quot charged regions o pH of mobile phase determine how charged protein is pH to pl I pH gt pl neg charge I pH pl 0 charge I pH lt pl pos charge o salts used to elute proteins off column by replacement Polymer lneaals with ampglt i39H39El F lltvargvIazli flmnctiumiall grniutn Friateiin m39imtum is azddxedl in Iznaliumrrl uuntaairuirIg1 quot ca39tiiun IEll39iI39IaF39IQtI39quotlli5 I salt ions compete with protein for binding to matrix letermi 39 I 2 39 g A I 39li1Ii 0 Size Exclusion Chromatography gel filtration size a 3F39f a39IiEIlTIlE mu ire il1mIlugl1 the column at irate I39IIE l39lEl39l f39E at the l P fl i Tlll llll I ribo o Proteins are hindered by matrix according to their size amp shape I Large proteins pass through easily small proteins H get stuckin matrix wk P fr Ff EIquotI Elf39I of f F interest 0 Affinity Chromatography binding affinity to matrix o Protein affinity is measured with accordance to ligand linked to matrix I o Free ligand is used to compete off bound protein of P H s X 39 interest l3939ii3939limture 5olluiticin V nif pmtneiins 39 I iiifliiiganizl L 0 Nickel Chromatography specific affinity chroma A K j o Uses nonnative engineered recombinant plasmid DNA encodes 612 His residues N or C terminus of protein of Vi interest 0 Engineered His binds to Ni2 in resin bound to surface 6 Elute with high concentration of imidazole y 0 Because imidazole is very similar to His competes Pmtein mmmei dd dj p P for bonding spot with Ni2 when transient HisNi 39quot quotquotquot 39quotquot 39 quot9 i l If a piilymeir lbnuind ii i yquotw 3 bond breaks lliigaind 5pEiiil lE fur 1 Le a gl s c y pr39 tEin afinterest Ii 2 3 4 5 3 4 5 E E B I Once imidazole takes up all the His binding spots unwanted Pmtieinsi Frmil afintmst are W 5i liEEll thriciugliii sis eluted by ligand then the protein of interest is freed column sizilutiiun 0 lmmunoaffinity Chromatography specific affinity chroma o Antibody coupled to beads o Protein interacts with antibody o Elute with LOW pH u 0 Protein elution is measurable through UV detection of protein content of elute o Proteins absorb UV light 280 nm Tyr Trp Phe aromatic residues o Plot absorbance vs elution volume UV absorbarice M L 5 PROTEIN ANALYTIC TECHNIQUES g 5 0 i ElLllIiDIquotI iiolumie Sammie M electrophoresis C quotquotquoti39iquot39 0 Separate proteins according to migration through gel matrix in electric field A Dimtiiun 0 Use SDS sodium dodecyl sulfate as ionic detergent that denatures proteins misraticn and confers NEGATIVE charge 0 Separate proteins according to size amp charge o Large proteins travel slower negative charged travel to positive end 39 39i a EC Elf 35 II SDSPAGE 0 Separate proteins based on size small proteins travel through gel faster 0 SDS breaks apart protein subunits llulol Weight Laclldelr 1 Z Myosin 2 IlquotII It7JlfI BGaiacto idFase 1I1i l25D T Glycogen phosphorylalse b Q7 flD I lBovine serum ailbuuirniri 661200 T Dvailbluimin 45l Jl lD Carbonianlirydrase 31llIUD Soybean trypsiniinhislbitor 2lI5ElD fl Lysozyme Ii 4400 it Mr Uinlmowrri a39 standlorcls protein isoelectric focusing 0 Amphoytes have both acid and base characteristics 0 Creates a linear pH gradient across and electric field 0 Separates proteins according to pl logM 0 Proteins stop migrating at pH pl 2D gel electrophoresis Fiirgst dimension lsoeilectiritrs iiII39Illl5ii I1g 6 39v 1 3 u 1 J eere pl iI5iflg ii ii i ilg 1 23 J Ill i lsoeleeitriic Focusing gel is plated on SE5 po l391racrgrlamide ge Eerzood F 9 rIlimersion F I EDS gpol39yrerir3rlami e 39 gel electrophoresis quotquot quot39 3 I Ieoreesiogi F ii Mr 1 Urn known protein Relatiire migration Ari emoholiyte 3 A g ftji V solution is pli 9 1amp3 iimoriporatjecl A l i into a gel her 39 g N E 539ae E 1 E c 39 N p p E p 39 E Equots 39 E9 F V 3 PH 3 i is J39 RHquotyiquotF r EL 5tallre pTHl greoliient Proteini soluitiolrl is Aflielr staiinirigg proteiirls is estahllilshetl il139lHl39IE added and electric are sl1IownVto ihe gel after eioplicetiioo liielrl is reoppllied distrio uteri ollong l lrl of an electric Iiielr g1ra dient aooorriin gi to their all uraluee 0 1 dimension isoelectric focusing 0 2 39 dimension SDSPAGE separate by MW Derzineosiisn g iii I a 5 J KEEPING TRACK OF 65 e i 0 39 Q In Q PROCESSES gJ Cg e E3 1 Bj 0 Specific activity proportion he j V f 039 E1 g339 3 gay o total protein in solution J3 Gg 5 gs 4r39 e we ya I A R that has enzyme activity of 4 J VKEQ quot gl zmlj H II1 g ll f ill l Si i 5 l 1I39 11quotEfquotquot3911 L1i 510 3Siiri r39 ri 14 it39ii Cl11ii111itiig1 iap11 Ci1 F I139 39IIEl11 C11139D11 1fl ID39gTl11411 1 D 1 Egg 1 W M T3 4 quotquotLff39fl il 39I39 quot W 0 f I I C l11quoto111ato g1 apl1quot quot 1 mm 50 100 20 PROTEIN STRUCTURE DETERMINATION Xray crystallography 0 PROCESS o Start with a very pure protein of interest and then form crystal lattice o shoot xrays at it o xrays bounce off and create diffraction pattern I diffraction pattern gives information on intensity and position of electrons I xrays are form of electromagnetic radiation made of sinusoidal oscillating magnetic and electric fields 0 amplitude half full width of oscillation of intensity 0 phase cl distance between origin and following maximum in wave distance 360 wavelength o use diffraction pattern to form electron density map o expressed as cl I atoms in molecule are surrounded by cloud of electrons 0 more electrons the bigger the cloud o determine density of electrons and use densities to locate actual atoms in protein I electron density equation summation of all the structure ie amplitude phases etc in a data set 0 structural amplitude F has to be measured relates to darknessintensity of spots phase cgt is not possible to measure directly 0 Diffraction pattern gives info on intensity and position 0 Allows for visualization of protein 3D structure Lets you o Understand biochemical properties and function I Ex reaction mechanism if it s an enzyme 0 PHASE PROBLEM o Diffraction pattern doesn39t tell us anything about phase of reflected xray o SOLUTIONS I Multiple lsomorphous Replacement MIR soak crystal in solution containing quotheavy atom which can change intensity of reflected X rays I Multiwavelength Anomalous Dispersion MAD uses heavy atom to change the wavelength of the X ray source 0 Heavy atom usually conferred by selenomethionine Molecular Replacement guess the phase of reflected X rays by using phases from similar structure solved previously NMR 0 HCNF and P have nuclear spins in a magnetic field 0 Can be measured in 1D and 2D plots 0 Data is used to computationally arrive at family of related structures that A 3 represent range of conformations consistent with measured NMR ZQ distance constraints if 0 Image is of 2D NMR spectrum diagonal line is equivalent to 1D NMR G 3 spectrum offdiagonal represents close interactions between atoms that E E might be distant in the 1D structure 5 A i 3 quotin H B tl I 5 ti I 475 I min I ELIE I fu high resolution cryo electron microscopy 397 1 hEquot aquot quotquotquotimquot quot quot39 0 Purify protein in vitreous ice not crystalline but more gelatinous makes sure that protein is not punctured during crystal formation and use electron beam to image the protein o Electron beam produces wavelength much shorter than light so very poor image resolution 0 Images provided are from different viewing angles so arrange pictures into classes depending on whether they are taken from similar angles or not LIGAND BINDING quantitation of ligand binding reveals aspects of protein function 0 Ligand typically a small molecule that binds to a macromolecule P L gt PL protein ligand complex ligands collide with target at rate k0 which depends on concentration units of sec391M391 o o ligands leave binding site at rate koff depends on strength of interaction units sec391 reactions that are strong have fewer dissociations per second ex 1O392sec reactions that are weak have more dissociations per second ex 106sec association constant Ka 0 K3 is a special case of the equilibrium constant Keq specifically used for a binding reaction 0 AG RTnKa L E K eq P L koff a dissociation constant Kd Measures strength of reaction K 1 PL d j K a PL Determined by measuring the fraction of protein bound protein binding sites occupied by ligand O L V H chemical 51l39Iif39t Ippmij 0 Fractional Binding 0 totalpmtein LKd 0 Given P L lt gt PL when G 05 O P L PL i0 O K 39 K3 PL 1 Fractionl a and Kd L of boijrlri 9 L protein 0395 L arbitrary units 0 GRAPHING o Plot of G is hyperbolic o K0 is ligand concentration at which 50 of ligand binding sites are occupied o Binding curve reveals Kd but experimental data must be fit to binding equation for hyperbola to accurately determine Kd I Scatchard Plot linearize data and plot data as straight line to determine K0 9 1 L 1 L Kd Kd S ope Kd 0 STEPS o Acquire binding data experimentally o Plot saturation curve 6 vs L 0 L and plot against E o Calculate Eiiniclling Curve Scatchard Plot L1 M 5 y 1 103000 12022 002 020 l2 005 03 0 01 05 3 02 074 E 6 04 004 4 00 003 2 10 000 U J 02 04 06 08 ll ll39lIn1l39Iu39 0 Higher K lower affinity weaker 0 Lower K higher affinity stronger o Lower L required to bind 2 of sites OXYGEN AND MYOGLOBIN Mb 0 Mb has 1 heme unit prosthetic group other chemical required to function o That 1 heme binds 1 02 since 02 is a gas graph 0 against partial pressure in killopascals 0 Curve suggests high affinity since nearly 100 is bound at low pO2 P150 g quot339 0 Graph is hyperbolic curve OXYGEN AND HEMOGLOBIN Hb 0 Need to bind oxygen in the lungs then release in tissues so it can be transferred to myoglobin o Have high affinity in lungs and low affinity in tissue 9 0 Change in Hb affinity is due to Hb binding cooperatively o Cooperativity determined by 4 structure o Affinity of Hb for 02 increases as more 02 is bound I High 02 is required for first 02 to bind then affinity increases 4 E W H 15 500fold for subsequent 02 binding events conformational change 1quot gm 30 in 4 structure p Klflhrfnqaf nily o Affinity decreases as less 02 is bound release in tissues M H We il3 l 39iquot i 39i5Ii I As 02 unloads affinity for remaining 02 decreases easier for all 02 39 quot mm to be released 9 04 o Graph for Hb is sigmoidal curve indicative of cooperativity M 1 lnwg niw A 5 1392 16 lF390 lliPal o Sigmoid curve is a hybrid of high and low affinity curves molecular mechanism for cooperativity conformational change O2 binding changes position of proximal His and Helix F Weak interactions between alpha and beta subunits help them slide past each other Interactions that stabilize Tstate are broken new interactions form that stabilize the R state Change in subunit conformation alters Fe2 conformation in other subunits drives them toward R state 02 has higher affinity for R state T state lower affinity stateDeoxy Hb R state higher affinity stateOxy Hb mu lll39I1 2


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