bi 421 midterm ii studyguide

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BI 421 Midterm II Study Guide 1

Afnity Chromatography Exploits Specifc Binding Behavior• Uses the proteins a!ility to !ind specifc molecules tightly"

!ut noncovalently• In a#nity chromatography" a molecule $ligand% hat

specifcally !inds to the protein o& interest is covalently

attached to an inert matrixo 'hen an impure protein solution is passed through this

chromatographic material" the desired protein !inds tothe immo!ili(ed ligand" )hereas other su!stances are)ashed through the column )ith the !u*er

• +he desired protein can !e recovered in highly purifed &orm!y changing the elution conditions to release the protein &romthe matrix

• ,dvantage- its a!ility to exploit the desired proteins uni.ue!iochemical properties rather than the small di*erences inphysicochemical properties !et)een proteins exploited !yother chromatographic methods

• Immunoa#nity chromatography / an anti!ody is attached tothe matrix in order to puri&y the protein against )hich theanti!ody )as raised

• Metal chelate a#nity chromatography / a divalent metal ion"such as 0n2 or i2 is attached to the chromatographic matrixso that proteins !earing metal3chelating groups $ie multiple5is side chains% can !e retained

o His tag 6 $5is%7 6 is attached to the 3 or 83terminus o&the polypeptide to !e isolated

o +his creates a metal ion3!inding site that allo)s therecom!inant protein to !e purifed !y metal chelatechromatography

o ,&ter the protein is eluted" 5is tag can !e removed !ythe action o& specifc protease )hose recognitionse.uence separates the $5is%7 se.uence &rom the rest o& the protein

9rotein Structure• 9rimary structure o& a protein is its linear se.uence o& amino acids• Secondary structure is the local spatial arrangement o& a

polypeptides !ac:!one atoms )ithout regard to the con&ormationso& its side chains

• +ertiary structure re&ers to the three3dimensional structure o& anentire polypeptide" including its side chains

• Many proteins are composed o& t)o or more polypeptide chains$su!units%;<uarternary structure re&ers to the spatial arrangemento& its su!units



= = = =


Secondary Structure• 9rotein secondary structure includes the regular polypeptide &olding

patterns" such as helices" sheets" and turns• +he peptide group has a rigid" planar structure as a conse.uence

o& resonance interactions that give the peptide !ond > 4?@ dou!le3!ond character-

• +his explanation is supported !y the o!servations that a peptidegroups 8; !ond is ?1A shorter than its ;8C single !ond andthat its 8D !ond is ??2 longer than that o& aldehydes and :etones

• Trans conormation: successive 8C atoms are on opposite sides o&the peptide Foining them

• Cis conormation: successive 8C atoms are on the same side o&the peptide !ond" is > :H・mol31 less sta!le than the transcon&ormation !ecause o& steric inter&erence !et)een neigh!oringside chains

• +orsion ,ngles !et)een 9eptide Groups escri!e 9olypeptide 8hain8on&ormations

o +he backbone or main chain o& aprotein re&ers to the atoms thatparticipate in peptide !onds" ignoring

the side chains o& the amino acidresidues

o +he con&ormation o& the !ac:!one canthere&ore !e descri!ed !y the torsionangles $dihedral anglesJrotationangles% around the 8C; !ond $K% andthe 8C;8 !ond $L% o& each residue



BI 421 Midterm II Study Guide Q

cleaved" either en(ymatically or chemically" to specifc&ragments that are small enough to !e se.uenced

• "roteases have side chain re.uirements &or theresidues Ran:ing the scissile peptide !ond $ie the !ondthat is to !e cleaved%

• +he digestive en(yme trypsin has the greatestspecifcity and cleaves peptide !onds on the 8 side o&the positively charged residues ,rg and ys i& the nextresidue is not 9ro

•

• , chemical reagent that promotes peptide !ond

cleavage at specifc residues / cyanogen bromide $8Br%" cleaves on the 8 side o& Met residues

8 Edman egradation =emoves a 9eptides Pirst ,mino ,cid=esidue

• nce peptide &ragments &ormed through specifccleavage reactions have !een isolated" their amino acidse.uences can !e determined

• #dman degradation




Mass Spectrometry etermines the Molecular Masses o&9eptides

• $ass spectrometry accurately measures the mass3to3charge $m/z % ratio &or ions in the gas phase $)here m isthe ions mass and z is its charge%

• #lectrospray ioni%ation &#'() techni.ueo , solution o& a macromolecule" such

a peptide" is sprayed &rom a narro)capillary tu!e maintained at high

voltage $>4??? T%" &orming fne" highcharged droplets &rom )hich thesolvent rapidly evaporates

o +his yields a series o& gas3phasemacromolecular ions +he charges result &rom the

protonation o& !asic side chainssuch as ,rg and ys

&*) phenylisothiocyanate &"(TC)reacts )ith the 3terminal aminogroup o& a polypeptide under mildal:aline conditions to &orm aphylthiocarbamyl &"TC) / !asiccondition

$2%+reat a!ove product )ithanhydrous tri+uoroacetic acid")hich cleaves the 3terminalresidue as a thia(olinonederivative !ut does not hydroly(eother peptide !onds

Edman degradation releasethe 3terminal amino acidresidue" !ut leaves intact threst o& the polypeptide chai

$A%+he thia(olinone3amino acid isselectively extracted into an organsolvent and is converted to the mosta!le phenylthiohydantoin $"Tderivative !y treatment )ith a.ueacid / acidic condition

Thus, it is possible to determine tamino acid sequence of a polypeptide chain from the N-terminus inward by subecting the

polypeptide to repeated cycles of



BI 421 Midterm II Study Guide V

o +he ions are directed into the massspectrometer" )hich measures theirm/z values )ith an accuracy o&>??1@

• Tandem mass spectrometry

o Short polypeptides $W2Q residues% can !e directlyse.uenced through the use o& a tandem massspectrometer

o Involves 2 mass spectrometers coupled in series$1%+he frst mass spectrometer &unctions to select

and separate the peptide ion o& interest &rompeptide ions o& di*erent masses as )ell as anycontaminants that may !e present

$2%+he selected peptide ion is then passed into acollision cell" )here it collides )ith chemically inertatoms such as He

$A%+he energy imparted to the peptide ion causes itto &ragment predominantly at only one o& itsseveral peptide !onds" there!y yielding one ort)o charged &ragments per original ion

$4%+he molecular masses o& the numerous charged&ragments so produced are then determined !ythe second mass spectrometer

#y comparing the molecular masses of successi"ely larger members of a family of fragments, the molecular masses and

therefore the identities of the corresponding amino acidresidues can be determined

+he Most 8ommon =egular Secondary Structures ,re the C 5elix and theN Sheet

• +he C 5elix is a coilo nly one polypeptide helix has !oth a &avora!le 53!onding

pattern and K and L values that &all )ithin the &ully allo)edregions o& the =amachandran diagram- the C 5elix

o iscovered !y ,inus "aulingo +he C helix is right-handed



BI 421 Midterm II Study Guide

o +he C 5elix has .6 residues per turn and a pitch $thedistance the helix rises along its axis per turn o& /.0 1

o +he C helices o& proteins have an average length o& > 12residues

o In the C helix" the !ac:!one 53!onds are arranged such that

the peptide 8D !ond o& the nth

residue points along the helixaxis to)ard the peptide ;5 group o& the $n4%th residueo ,mino acid side chains proFect out)ard and do)n)ard &rom

the helix;avoiding steric inter&erence )ith the polypeptide!ac:!one and )ith each other

9ropensities 234 C 5elix$=3groups that love to !e in C

5elix%

9ropensities A5A('T C5elix

Ala7 5lu7 5ln7 ,eu7 $et "ro7 5ly

• N Sheets ,re Pormed &rom Extended 8hainso i:e the C 5elix" the N Sheet uses the &ull 53!onding capacity

o& the polypeptide !ac:!oneo $owe"er, in % &heets, $-bonding occurs between neighboring

polypeptide chains rather than within one as in an ' $eli( o +here are 2 types o& sheets

1 Antiparallel 8 'heet ; neigh!oring 53!ondedpolypeptide

chains run in opposite directions

2 "arallel 8 'heet ; 53!onded chains extend in the

same direction



BI 421 Midterm II Study Guide X

o 9arallel N Sheets are less sta!le than antiparallel N Sheet"possi!ly !ecause the 53!onds o& parallel sheets are distortedcompared to those o& the antiparallel sheets

o N Sheet exhi!it a pronounced right-handed t9ist )henvie)ed along their polypeptide strands

o +he geometry o& a particular N Sheet is a compromise!et)een optimi(ing the con&ormational energies o& itspolypeptide chains and preserving its 53!onding

• +urns 8onnect Some Units o& Secondary Structureo 9olypeptide segments )ith regular secondary structure such

as C helices or the strands o& N Sheets are o&ten Foined !ystretches o& polypeptide that a!ruptly change direction

o 4eerse turns or 8 bends $so named !ecause they o&tenconnect successive strands o& antiparallel N Sheet% al)aysoccur at protein sur&aces usually involve 4 successive aminoacid residues arranged in one o& t)o )ays" Type ( and Type((" that di*er !y a 1?Y Rip o& the peptide unit lin:ing residue2 and A

Both turns are sta!ili(ed !y 53!onds



BI 421 Midterm II Study Guide 1?

9ropensities 234 N Sheets$=3groups that love to !e in N

Sheets%

9ropensities A5A('T NSheets

;al7 (le7 "he7 Trp7 Tyr "ro

• Pi!rous 9roteins 5ave =epeating Secondary Structureso 9roteins can !e classifed as <brous or globular

Examples o& f!rous proteins;keratin and collagen

o C Zeratin is a 8oiled 8oil =eratin is a mechanically dura!le and relatively

unreactive protein that occurs in all higher verte!rates Zeratins have !een classi&ied as either C :eratins

$mammals% or N :eratins $!irds [ reptiles% > keratin

\3ray di&&raction pattern o& C :eratin resem!les Chelix

Exhi!its Q1 spacing rather than Q4 distancecorresponding to the pitch o& the C helix

Two ' )eratin polypeptides, each of which formsan ' heli(, twisting around each other form a left-handed coil $coiled coil structure%

• +he central >A1?3residue segment o& eachpolypeptide chain has a V3residue pseudorepeat"

a-b-c-d-e-f-g" )ith nonpolar residues at position a and d

• C :eratin is rich in 8ys residues" )hich &ormdisulfde !onds that cross3lin: adFacentpolypeptide chains $sta!ili(ation%

• +reating hair !y curlingJstraightening a*ects thedisul&ide !onds in the C :eratin $apply oxidi(ingagent to re3esta!lish disulfde !onds%




.ariations in amino acid sequence as well as the o"erall

structure of the folded protein can distort the regularconformations of secondary structural elements

8 bulge 6 a strand o& polypeptide in a N sheet maycontain an ]extra^ residue that is not 53!onded to a

neigh!oring strand producing a distortion $N !ulge%• ex 9ro residue produces a ]:in:^ in an C helix or

N sheet

• +ertiary Structures 8ontain 8om!inations o& Secondary Structureo 8ertain 8om!inations o& Secondary Structure Porm Moti&s

Groupings o& secondary structural elements;supersecondary structures

1 8>8> structure &4ossman 2old);an C helixconnects t)o parallel strands o& a N sheet

2 888 &8 hairpin);consists o& antiparallel strands

connected !y relatively tight reverse turnsA >>> &> structure);t)o successive antiparallel Chelices pac: against each other )ith their axesinclined

o Most 9roteins 8an Be 8lassi&ied as C" N" or CJN +he maFor types o& secondary structural elements occur

in glo!ular proteins in varying proportions andcom!inations

• > proteins;proteins that consist only o& Chelices spanned !y short connecting lin:s

• 8 proteins;proteins that contain a large

proportion o& N sheets and are devoid o& C helices• >?8 proteins;proteins that largely consist o&

mixtures o& !oth types o& secondary structure$>A1@ C helix and >2@ N sheet%

8 barrels >?8 barrel

• Most 9rotein Structures ,re etermined !y \3=ay 8rystallography oruclear Magnetic =esonance

o @-ray crystallography ; a techni.ue that directly imagesmolecules

\3rays are used !ecause the uncertainty in locating ano!Fect is approximately e.ual to the )avelength o& theradiation used to o!serve it $covalent !ond distancesand the )avelengths o& the \3rays used in structuralstudies are !oth > *./ 1%

, crystal o& the molecule to !e imaged is exposed to acollimated !eam o& \3rays and the resulting diraction



BI 421 Midterm II Study Guide 1A

pattern" )hich arises &rom the regularly repeatingpositions o& atoms in the crystal" is recorded !y aradiation detector

\3=ays interact exclusively )ith the electrons in matter"not the nuclei;there&ore" x3ray structure is an image o&the electron density o& the molecule under study

•

#lectron density maps are presented )ith theaid o& computer graphs as one or more sets o&contours" in )hich a contour represents a specifclevel o& electron density

o Most 9rotein 8rystal Structures Exhi!it ess than ,tomic=esolution

9rotein crystals di*er &rom those o& most small organicand inorganic molecules in !eing highly hydrated $4?37?@ )ater !y volume%

• +he a.ueous solvent o& crystalli(ation is necessary

&or the structural integrity o& the protein crystals"!ecause )ater is re.uired &or the structuralintegrity o& native proteins themselves

8rystals have a resolution limit o& their si(e• 9rotein crystals typically have resolution limits in

the range 1Q to A? $1Q resolution is veryclear" A? resolution is not very clear%

\3ray di*raction pattern-

+he intensities o& the di*ractionmaxima $dar:ness o& spots% arethe electron densities o& eachatom

3 Intensities are due to )aves




Most protein crystal structures are too poorly resolved&or their electron density maps to reveal clearly thepositions o& individual atoms;nevertheless" thedistinctive shape o& the polypeptide !ac:!one permits itto !e traced )hich allo)s the positions and orientations

o& its side chains to !e deduced• , protein structure cannot !e elucidated &rom its

electron density map alone" !ut :no)ing theprimary structure o& the protein permits these.uence o& amino acid residues to !e ftted tothe electron density map

o Most 8rystalline 9roteins Maintain +heir ative 8on&ormations 8rystalline proteins assume very nearly the same

structures that they have in solution

o 9rotein Structures 8an Be etermined !y M= uclear magnetic resonance &$4) is the

o!servation that an atomic nucleus $proton% resonatesin an applied magnetic feld in a )ay that is sensitive toits electronic environment and its interactions )ithnear!y nuclei

T9o-dimensional &B) $4 spectroscopy ; yieldsadditional pea:s arising &rom the interactions o& protonsthat are less than Q apart $radiate at one _ o&radiation;!rea:;radiate again%

• Correlation spectroscopy &C3'D) provides

interatomic distances !et)een protons that arecovalently connected through one or t)o otheratoms" such as the 5 atoms attached to the and8C o& the same amino acid $K torsion angle%

• uclear 3erhauser spectroscopy &3#'D) provides interatomic distances &or protons thatare close in space although they may !e &ar apartin the protein se.uence

Interatomic distance measurements are used tocompute the proteins A3 structure

5o)ever" since interproton distance measurements are

imprecise" they cannot imply a uni.ue structure !utrather" a consistent )ith an ensem!le o& closely relatedstructures

8onse.uently" an M= structure o& a protein is o&tenpresented as a sample

• Side 8hain ocation Taries )ith 9olarity



BI 421 Midterm II Study Guide 1Q

o ,mino acid side chains in glo!ular proteins are spatiallydistri!uted according to their polarities

on3polar residues ;al7 ,eu7 (le7 $et7 and "he occurmostly in the interior o& a protein $hydropho!ic%

8harged polar residues Arg7 His7 ,ys7 Asp7 and 5lu

are located on the sur&ace o& the protein $hydrophilic% Uncharged polar groups 'er7 Thr7 Asn7 5ln7 and Tyr are

located on the protein sur&ace !ut also occur in theinterior o& the molecule

<uaternary Structure and Symmetry• Euaternary 'tructure;the spatial arrangement o& polypeptide

su!units• In the case o& en(ymes" increasing a proteins si(e tends to !etter

fx the A3 positions o& its reacting groupso Increasing the size of an enzyme through the association of

identical subunits is more ecient than increasing the lengthof its polypeptide chain since each subunit has an acti"e site+ore importantly, the subunit construction of many enzymes pro"ides the structural basis for the regulation of theiracti"ities

• Su!units Usually ,ssociate oncovalentlyo , multisu!unit protein may consist o& identical or nonidentical

polypeptide chains $ie 5emoglo!in C2N2%o The contact regions between subunits resemble the interior of

a single-subunit protein0 they contain closely pac)ed nonpolar side chains, $-bonds in"ol"ing the polypeptide bac)bones andtheir side chains and interchain disul1de bonds

• Su!units ,re Symmetrically ,rrangedo 9roteins cannot have inversionJmirror symmetry;proteins can

have only rotational symmetryo Cyclic symmetry;identical su!units are related !y a single

axis o& rotationo ihedral symmetry;generated )hen n3&old rotation axis

intersects a 23&old rotation axis at right angles

9rotein Sta!ility• ative proteins are only marginally sta!le under physiological

conditions

General 9roperties o& En(ymes• En(ymes




1 Higher reaction rates;the rates o& en(ymatically cataly(edreactions are typically 1?7 to 1?12 times greater than those o&the corresponding uncataly(ed reactions

2 $ilder reaction conditions;en(ymatically cataly(edreactions occur under relatively mild conditions- temperatures

!elo) 1??Y8" atmospheric pressure" and nearly neutral p5A 5reater reaction speci<city;en(ymes have a vastlygreater degree o& specifcity )ith respect to the identities o&!oth their substrates and their products

4 Capacity or regulation;the catalytic activities o& manyen(ymes vary in response to the concentrations o& su!stancesother than their su!strates

• En(ymes ,re 8lassifed !y the +ype o& =eaction +hey 8ataly(eo En(ymes are commonly named !y appending the su#x 6ase

to the name o& the en(ymes su!strate or to a phrasedescri!ing the en(ymes catalytic action $ie alcohol

dehydrogenase%o En(ymes are classifed and named according to the nature o&

the chemical reactions they cataly(eo +here are 7 maFor classes o& en(ymatic reactions

1 xidoreductases $redox%2 +rans&ormasesA 5ydrolases4 yases $&orms 838 !onds%Q Isomerases7 igases $!ond &orming%

o Each en(yme is assigned t)o names and a &our3partclassifcation num!er

Triial name is convenient &or everyday use 'ystematic name is the name o& its su!strate$s%

&ollo)ed !y a )ord ending in 6ase speci&ying the type o&reaction the en(yme cataly(es according to its maForgroup classifcation

#C classi<cation• En(ymes ,ct on Specifc Su!strates

o In general" a su!strate3!inding site consists o& an indentation

or cle&t on the sur&ace o& an en(yme molecule that iscomplementary in shape to the su!strate $geometriccomplementarity%

o Moreover" the amino acid residues that &orm the !inding siteare arranged to specifcally attract the su!strate $electroniccomplementarity%



BI 421 Midterm II Study Guide 1V

o \3ray studies indicate that the su!strate3!inding sites o& mosten(ymes are largely pre&ormed !ut undergo somecon&ormational change on su!strate !inding $induced <t%

• En(ymes ,re Stereospecifco En(ymes are highly specifc !oth in !inding chiral su!strates

and in cataly(ing their reactionso 'tereospeci<cty arises !ecause en(ymes &orm asymmetric

active siteso Nearly all enzymes that participate in chiral reactions are

absolutely stereospeci1c

• En(ymes Tary in Geometric Specifcityo , su!stance o& the )rong chirality )ill not ft productively into

an en(ymatic !inding siteo Most en(ymes are .uite selective a!out the identities o& the

chemical groups on their su!strates $geometric speci<city%• Some En(ymes =e.uire 8o&actors

o ,lthough en(ymes can cataly(e oxidation3reduction reactions"they can do so only in association )ith small coactors

o 8o&actors may !e metal ions" such as 8u2" PeA" or 0n2

o 8o&actors may also !e organic molecules :no)n ascoen%ymes

o Some co&actors are only transiently associated )ith a givenen(yme molecule" so that they &unction as cosubstrates $ie, / ,5%

o "rosthetic groups are permanently associated )ith theirprotein" o&ten !y covalent !onds




o , catalytically active en(yme3co&actor complex;holoen%ymeo En(ymatically inactive protein resulting &rom the removal o& a

holoen(ymes co&actor;apoen%yme

o

o 8oen(ymes must !e regenerated 8oen(ymes are chemically changed !y the en(ymatic

reactions in )hich they participate In order to complete the catalytic cycle, the coenzyme

must return to its original state

• Por transiently !ound coen(ymes $cosu!strate%"the regeneration reaction may !e cataly(ed !y adi*erent en(yme

• Por prosthetic groups" regeneration occurs as parto& the en(yme reaction se.uenceo

•

,ctivation Energy and the =eaction 8oordinateo Transition state theory 8onsider a !imolecular reaction involving A atoms-o

+he point o& highest &ree energy;transition state

Transition state diagram?reaction coordinatediagram• +he reactants and products are states o& minimum

&ree energy" and the transition state correspondsto the highest point o& the diagram

F5G &ree energy o actiation)the &reeenergy o& the transition state less that that o& thereactants

The greater the value of F5G7 the slower thereaction rate



BI 421 Midterm II Study Guide 1X

o




o Ultracentri&ugation Separates Macromolecules !y Mass• 3nly when subected to enormous accelerations do

macromolecules begin to sediment, much li)e grains of sandin water

• Iltracentriuge;developed !y 'edberg;demonstrated

that proteins are macromolecules )ith homogenouscompositions and that many proteins contain su!units

• The rate at which a particle sediments in the

ultracentrifuge is related to its mass $the density o& thesolution and the shape o& the particle a*ects thesedimentation rate%

• , proteins sediment coefcient $its sedimentation velocityper unity o& centri&ugal &orce% is expressed in units o& 1?31As;'edbergs &')

• +he relationship !et)een molecular mass and sedimentationcoe#cient is not linearb there&ore" values o& sedimentationcoe#cients are not additive

o +he sedimentation coe#cients o& proteins range &rom1S to Q?S

o

•

Ultracentri&ugation is use&ul &or &ractioning macromoleculeso Sedimentation is carried out in a solution o& an inertsu!stance in )hich the concentration" and there&ore thedensity" o& the solution increases &rom the top to the!ottom o& the centri&uge tu!e

o Such density gradients can !e generated !y flling thecentri&uge tu!e )ith layers o& sucrose solutions o&




decreasing concentrations )ith the sample o&macromolecules on the very top

o uring centri&ugation" each species o& macromoleculemoves through the pre&ormed gradient at a rate largelydetermine !y its sedimentation coe#cient and travels

as a (one that can !e separated &rom other (oneso ,&ter centri&ugation" the tu!e is punctured to collect

&ractions containing the separated macromolecules

bi 421 midterm ii studyguide

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