Page23

Chapter2
ProteinsI:
CompositionandStructure
RichardM.SchultzandMichaelN.Liebman

2.1FunctionalRolesofProteinsinHumans 24

2.2AminoAcidCompositionofProteins 25

ProteinsArePolymersofa AminoAcids 25

CommonAminoAcidsHaveaGeneralStructure 25

SideChainsDefineChemicalNatureandStructuresofDifferentAmino 25
Acids

AminoAcidsHaveanAsymmetricCenter 28

AminoAcidsArePolymerizedintoPeptidesandProteins 28

CystineIsaDerivedAminoAcid 30

2.3ChargeandChemicalPropertiesofAminoAcidsandProteins 30

IonizableGroupsofAminoAcidsandProteinsAreCriticalforBiological 30
Function

IonicFormofanAminoAcidorProteinCanBeDeterminedataGiven 32
pH

TitrationofaMonoaminoMonocarboxylicAcid:Determinationofthe 32
IsoelectricpH

TitrationofaMonoaminoDicarboxylicAcid 33

GeneralRelationshipbetweenChargePropertiesofAminoAcidsand 34
ProteinsandpH

AminoAcidsandProteinsCanBeSeparatedBasedonpIValues 35

AminoAcidSideChainsHavePolarorApolarProperties 38

AminoAcidsUndergoaVarietyofChemicalReactions 38

2.4PrimaryStructureofProteins 39

2.5HigherLevelsofProteinOrganization 42

ProteinsHaveaSecondaryStructure 43

a HelicalStructure 43

b Structure 44

SupersecondaryStructures 44

ProteinsFoldintoaThreeDimensionalStructureCalledtheTertiary 44
Structure

HomologousThreeDimensionalDomainStructuresAreOftenFormed 47
fromCommonArrangementsofSecondaryStructures

AQuaternaryStructureOccursWhenSeveralPolypeptideChainsForm 48
aSpecificNoncovalentAssociation

2.6OtherTypesofProteins 49

FibrousProteinsIncludeCollagen,Elastin,a Keratin,and 50
Tropomyosin

DistributionofCollageninHumans 50

AminoAcidCompositionofCollagen 50

AminoAcidSequenceofCollagen 50

StructureofCollagen 52

FormationofCovalentCrosslinksinCollagen 54

ElastinIsaFibrousProteinwithAllysineGeneratedCrosslinks 54

a KeratinandTropomyosin 55

LipoproteinsAreComplexesofLipidswithProteins 56

GlycoproteinsContainCovalentlyBoundCarbohydrate 60

TypesofCarbohydrateProteinCovalentLinkages 61

2.7FoldingofProteinsfromRandomizedtoUniqueStructures:Protein 62
Stability

TheProteinFoldingProblem:APossiblePathway 62

ChaperoneProteinsMayAssisttheProteinFoldingProcess 63

NoncovalentForcesLeadtoProteinFoldingandContributetoa 63
Protein'sStability

HydrophobicInteractionForces 64

HydrogenBonds 66

ElectrostaticInteractions 66

VanderWaalsLondonDispersionForces 66

DenaturationofProteinsLeadstoLossofNativeStructure 67

2.8DynamicAspectsofProteinStructure 68

2.9MethodsforCharacterization,Purification,andStudyofProtein 69
StructureandOrganization

SeparationofProteinsonBasisofCharge 69

CapillaryElectrophoresis 70

SeparationofProteinsBasedonMolecularMassorSize 71

Ultracentrifugation:DefinitionofSvedbergCoefficient 71

MolecularExclusionChromatography 72

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PolyacrylamideGelElectrophoresisinthePresenceofaDetergent 72

HPLCChromatographicTechniquesSeparateAminoAcids,Peptides, 72
andProteins

AffinityChromatography 73

GeneralApproachtoProteinPurification 73

DeterminationofAminoAcidCompositionofaProtein 74

TechniquestoDetermineAminoAcidSequenceofaProtein 74

XRayDiffractionTechniquesAreUsedtoDeterminetheThree 76
DimensionalStructureofProteins

VariousSpectroscopicMethodsAreEmployedinEvaluatingProtein 79
StructureandFunction

UltravioletLightSpectroscopy 79

FluorescenceSpectroscopy 79

OpticalRotatoryDispersionandCircularDichroismSpectroscopy 80

NuclearMagneticResonance 81

Bibliography 82

QuestionsandAnswers 83

ClinicalCorrelations

2.1PlasmaProteinsinDiagnosisofDisease 37

2.2DifferencesinPrimaryStructureofInsulinsusedinTreatmentof 41
DiabetesMellitus

2.3ANonconservativeMutationOccursinSickleCellAnemia 42

2.4SymptomsofDiseasesofAbnormalCollagenSynthesis 50

2.5Hyperlipidemias 56

2.6Hypolipoproteinemias 59

2.7GlycosylatedHemoglobin,HbA1c 62

2.8UseofAminoAcidAnalysisinDiagnosisofDisease 74

2.1
FunctionalRolesofProteinsinHumans

Proteinsperformasurprisingvarietyofessentialfunctionsinmammalianorganisms.Thesemaybegroupedintodynamicandstructural.Dynamicfunctionsinclude
transport,metaboliccontrol,contraction,andcatalysisofchemicaltransformations.Intheirstructuralfunctions,proteinsprovidethematrixforboneandconnective
tissue,givingstructureandformtothehumanorganism.

Animportantclassofdynamicproteinsaretheenzymes.Theycatalyzechemicalreactions,convertingasubstratetoaproductattheenzyme'sactivesite.Almostallof
thethousandsofchemicalreactionsthatoccurinlivingorganismsrequireaspecificenzymecatalysttoensurethatreactionsoccurataratecompatiblewithlife.The
characterofanycellisbasedonitsparticularchemistry,whichisdeterminedbyitsspecificenzymecomposition.Genetictraitsareexpressedthroughsynthesisof
enzymes,whichcatalyzereactionsthatestablishthephenotype.Manygeneticdiseasesresultfromalteredlevelsofenzymeproductionorspecificalterationstotheir
aminoacidsequence.Transportisanothermajorfunctionforproteins.Particularexamplesdiscussedingreaterdetailinthistextarehemoglobinandmyoglobin,which
transportoxygeninbloodandinmuscle,respectively.Transferrintransportsironinblood.Transportproteinsbindandcarrysteroidhormonesinbloodfromtheirsite
ofsynthesistotheirsiteofaction.Manydrugsandtoxiccompoundsaretransportedboundtoproteins.Proteinsparticipateincontractilemechanisms.Myosinand
actinfunctioninmusclecontraction.

Proteinshaveaprotectiverolethroughacombinationofdynamicfunctions.Immunoglobulinsandinterferonareproteinsthatprotectthehumanagainstbacterialor
viralinfection.Fibrinstopsthelossofbloodoninjurytothevascularsystem.

Manyhormonesareproteinsorpeptides.Proteinhormonesincludeinsulin,thyrotropin,somatotropin(growthhormone),luteinizinghormone,andfolliclestimulating
hormone.Manydiversepolypeptidehormoneshavealowmolecularweight(<5000)andarereferredtoaspeptides.Ingeneral,thetermproteinisusedfor
moleculescomposedofover50aminoacidsandthetermpeptideisusedformoleculesoflessthan50aminoacids.Importantpeptidehormonesinclude
adrenocorticotropinhormone,antidiuretichormone,glucagon,andcalcitonin.

Proteinscontrolandregulategenetranscriptionandtranslation.TheseincludehistonesthatarecloselyassociatedwithDNA,repressorandenhancertranscription
factorsthatcontrolgenetranscription,andproteinsthatformapartoftheheteronuclearRNAparticlesandribosomes.

 Page25

Structuralproteinsfunctionin"brickandmortar"roles.Theyincludecollagenandelastin,whichformthematrixofboneandligamentsandprovidestructuralstrength
andelasticitytoorgansandthevascularsystem.a Keratinformsthestructureofepidermaltissue.

Anunderstandingofboththenormalfunctioningandthepathologyofthemammalianorganismrequiresaclearunderstandingofthepropertiesoftheproteins.

2.2
AminoAcidCompositionofProteins

ProteinsArePolymersofa AminoAcids

Itisnotablethatallthedifferenttypesofproteinsareinitiallysynthesizedaspolymersofonly20aminoacids.Thesecommonaminoacidsaredefinedasthosefor
whichatleastonespecificcodonexistsintheDNAgeneticcode.Thereare20aminoacidsforwhichDNAcodonsareknown.Transcriptionandtranslationofthe
DNAcoderesultinpolymerizationofaminoacidsintoaspecificlinearsequencecharacteristicofaprotein(Figure2.1).Inadditiontothecommonaminoacids,
proteinsmaycontainderivedaminoacids,whichareusuallyformedbyanenzymefacilitatedreactiononacommonaminoacidafterthataminoacidhasbeen
incorporatedintoaproteinstructure.Examplesofderivedaminoacidsarecystine(seep.30),desmosineandisodesmosinefoundinelastin,hydroxyprolineand
hydroxylysinefoundincollagen,and carboxyglutamatefoundinprothrombin.

Figure2.1
Geneticinformationistranscribed
fromaDNA
sequenceintomRNAandthentranslatedto
theaminoacidsequenceofaprotein.

CommonAminoAcidsHaveaGeneralStructure

CommonaminoacidshavethegeneralstructuredepictedinFigure2.2.Theycontainincommonacentralalpha(a )carbonatomtowhichacarboxylicacidgroup,
anaminogroup,andahydrogenatomarecovalentlybonded.Inaddition,thea carbonatomisboundtoaspecificchemicalgroup,designatedRandcalledtheside
chain,thatuniquelydefineseachofthe20commonaminoacids.Figure2.2depictstheionizedformofacommonaminoacidinsolutionatpH7.Thea aminogroup
isprotonatedandinitsammoniumionformthecarboxylicacidgroupisinitsunprotonatedorcarboxylateionform.

SideChainsDefineChemicalNatureandStructuresofDifferentAminoAcids

StructuresofthecommonaminoacidsareshowninFigure2.3.Alkylaminoacidshavealkylgroupsidechainsandincludeglycine,alanine,valine,leucine,and
isoleucine.Glycinehasthesimpleststructure,withR=H.Alaninecontainsamethyl(CH3)sidechaingroup.ValinehasanisopropylRgroup(Figure2.4).The
leucineandisoleucineRgroupsarebutylgroupsthatarestructuralisomersofeachother.Inleucinethebranchingintheisobutylsidechainoccursonthegamma(g)
carbonoftheaminoacid.Inisoleucineitisbranchedatthebeta(b )carbon.

Thearomaticaminoacidsarephenylalanine,tyrosine,andtryptophan.ThephenylalanineRgroupcontainsabenzenering,tyrosinecontainsaphenolgroup,andthe
tryptophanRgroupcontainstheheterocyclicstructure,indole.

Figure2.2
General
structure
ofthe
common
amino
acids.

 Page26

Figure2.3
Structuresofthecommonaminoacids.ChargeformsarethosepresentatpH7.0.

Figure2.4
Alkylsidechainsofvaline,leucine,andisoleucine.

 Page27

Figure2.5
Sidechains
ofaspartate
andglutamate.

Ineachcasethearomaticmoietyisattachedtothea carbonthroughamethylene(CH2)carbon(Figure2.3).

Sulfurcontainingcommonaminoacidsarecysteineandmethionine.Thecysteinesidechaingroupisathiolmethyl(HSCH2).Inmethioninethesidechainisa
methylethylthiolether(CH3SCH2CH2).

Therearetwohydroxy(alcohol)containingcommonaminoacids,serineandthreonine.Theserinesidechainisahydroxymethyl(HOCH2).Inthreonineanethanol
structureisconnectedtothea carbonthroughthecarboncontainingthehydroxylsubstituent,resultinginasecondaryalcoholstructure(CH3CHOHCHa).

Figure2.6
Guanidiniumandimidazoliumgroups
ofarginineandhistidine.

Theprolinesidechainisuniqueinthatitincorporatesthea aminogroup.Thusprolineismoreaccuratelyclassifiedasana iminoacid,sinceitsa amineisa

secondaryaminewithitsa nitrogenhavingtwocovalentbondstocarbon(tothea carbonandsidechaincarbon),ratherthanaprimaryamine.Incorporationofthe
a aminonitrogenintoafivememberedringconstrainstherotationalfreedomaroundtheNaCabondinprolinetoaspecificrotationalangle,whichlimits
participationofprolineinpolypeptidechainconformations.

TABLE2.1AbbreviationsfortheAmino
Acids

Abbreviation

Three One
AminoAcid Letter Letter

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic Asp D

Asparagineor Asx B
aspartic

Cysteine Cys C

Glycine Gly G

Glutamine Gln Q

Glutamic Glu E

Glutamineor Glx Z
glutamic

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

TheaminoacidsdiscussedsofarcontainsidechainsthatareunchargedatphysiologicalpH.Thedicarboxylicmonoaminoacidscontainacarboxylicgroupintheir
sidechain.Aspartatecontainsacarboxylicacidgroupseparatedbyamethylenecarbon(CH2)fromthea carbon(Figure2.5).Inglutamate(Figure2.5),the
carboxylicacidgroupisseparatedbytwomethylene(CH2CH2)carbonatomsfromthea carbon(Figure2.2).AtphysiologicalpH,sidechaincarboxylicacid
groupsareunprotonatedandnegativelycharged.Dibasicmonocarboxylicacidsincludelysine,arginine,andhistidine(Figure2.3).Inthesestructures,theRgroup
containsoneortwonitrogenatomsthatactasabasebybindingaproton.ThelysinesidechainisaNbutylamine.Inarginine,thesidechaincontainsaguanidino
group(Figure2.6)separatedfromthea carbonbythreemethylenecarbonatoms.Boththeguanidinogroupofarginineandthee aminogroupoflysineare
protonatedatphysiologicalpH(pH~7)andintheirchargedform.Inhistidinethesidechaincontainsafivememberedheterocyclicstructure,theimidazole(Figure
2.6).The oftheimidazolegroupisapproximately6.0inwaterphysiologicalsolutionscontainrelativelyhighconcentrationsofbothbasic(imidazole)andacidic
(imidazolium)formsofthehistidinesidechain(seeSection2.3).

Thelasttwocommonaminoacidsareglutamineandasparagine.Theycontainanamidemoietyintheirsidechain.Glutamineandasparaginearestructuralanalogs
ofglutamicacidandasparticacidwiththeirsidechaincarboxylicacidgroupsamidated.UniqueDNAcodonsexistforglutamineandasparagineseparatefromthose
forglutamicacidandasparticacid.TheamidesidechainsofglutamineandasparaginecannotbeprotonatedandareunchargedatphysiologicalpH.

Inordertorepresentthesequenceofaminoacidsinaprotein,threeletterandoneletterabbreviationsforthecommonaminoacidshavebeenestablished(Table2.1).
Theseabbreviationsareuniversallyacceptedandwillbeused

 Page28

throughoutthebook.Thethreeletterabbreviationsofasparticacid(Asp)andglutamicacid(Glu)shouldnotbeconfusedwiththoseforasparagine(Asn)and
glutamine(Gln).Inexperimentallydeterminingtheaminoacidsofaproteinbychemicalprocedures,onecannoteasilydifferentiatebetweenAsnandAsp,orbetween
GlnandGlu,becausethesidechainamidegroupsinAsnandGlnarehydrolyzedandgenerateAspandGlu(seeSection2.9).Inthesecases,thesymbolsofAsxfor
AsporAsn,andGlxforGluorGinindicatethisambiguity.AsimilarschemeisusedwiththeoneletterabbreviationstosymbolizeAsporAsn,andGluorGln.

Figure2.7
Absoluteconfigurationofanaminoacid.

AminoAcidsHaveanAsymmetricCenter

ThecommonaminoacidswiththegeneralstructureinFigure2.2havefoursubstituents(R,H,COO,NH3+)covalentlybondedtothea carbonatominthea amino

acidstructure.Acarbonatomwithfourdifferentsubstituentsarrangedinatetrahedralconfigurationisasymmetricandexistsintwoenantiomericforms.Thuseachof
theaminoacidsexhibitsopticalisomerismexceptglycine,inwhichR=Handthustwoofthefoursubstituentsonthea carbonatomarehydrogen.Theabsolute
configurationforanaminoacidisdepictedinFigure2.7usingtheFischerprojectiontoshowthedirectioninspaceofthetetrahedrallyarrangeda carbonsubstituents.
Thea COOgroupisdirectedupandbehindtheplaneofthepage,andtheRgroupisdirecteddownandbehindtheplaneofthepage.Thea Handa NH3+
groupsaredirectedtowardthereader.Anaminoacidheldinthiswayprojectsitsa NH3+groupeithertotheleftorrightofthea carbonatom.Byconvention,ifthe
a NH3+isprojectedtotheleft,theaminoacidhasanLabsoluteconfiguration.Itsopticalenantiomer,witha NH3+projectedtowardtheright,hasaDabsolute
configuration.InmammalianproteinsonlyaminoacidsofLconfigurationarefound.TheLandDdesignationsrefertotheabilitytorotatepolarizedlighttotheleft(L,
levo)orright(D,dextro)fromitsplaneofpolarization.Astheaminoacidsinproteinsareasymmetric,theproteinsthatcontainthemalsoexhibitasymmetric
properties.

Figure2.8
Peptidebondformation.

AminoAcidsArePolymerizedintoPeptidesandProteins

Polymerizationofthe20commonaminoacidsintopolypeptidechainsincellsiscatalyzedbyenzymesandisassociatedwiththeribosomes(Chapter15).Chemically,
thispolymerizationisadehydrationreaction(Figure2.8).Thea carboxylgroupofanaminoacidwithsidechainR1formsacovalentpeptidebondwiththea amino
groupoftheaminoacidwithsidechainR2byeliminationofamoleculeofwater.Thedipeptide(twoaminoacidresiduesjoinedbyasinglepeptidebond)canthen
formasecondpeptidebondthroughitsterminalcarboxylicacidgroupandthea aminoofathirdaminoacid(R3),togenerateatripeptide(Figure2.8).Repetitionof
thisprocessgeneratesapolypeptideorproteinofspecificaminoacidsequence(R1R2R3R4Rn).Theaminoacidsequenceofthepolypeptidechainsisthe
primarystructureoftheprotein,anditispredeterminedbytheDNAsequenceofitsgene(Chapter14).Itistheuniqueprimarystructurethatenablesapolypeptide
chaintofoldintoaspecificthreedimensionalstructurethatgivestheproteinitschemicalandphysiologicalproperties.

Figure2.9
Electronicisomer
structuresofapeptidebond.

Apeptidebondcanberepresentedusingtworesonanceisomers(Figure2.9).InstructureI,adoublebondislocatedbetweenthecarbonylcarbonandcarbonyl
oxygen(C =O),andthecarbonylcarbontonitrogen(C N)linkageisasinglebond.InstructureII,thecarbonylcarbontooxygenbond(C O)isasinglebondand
thebondlocatedbetweenthecarbonylcarbonandnitrogenisadoublebond(C =N).InstructureIIthereisanegativechargeontheoxygenandapositivechargeon
thenitrogen.Actualpeptidebondsarea

 Page29

resonancehybridofthesetwoelectronisomerstructures,thecarbonylcarbontonitrogenbondhavinga50%doublebondcharacter.Thehybridbondissupported
byspectroscopicmeasurementsandXraydiffractionstudies,thelattershowingthatthecarbonylcarbontonitrogenpeptidebondlength(1.33)isapproximately
halfwaybetweenthatfoundforaCNsinglebond(~1.45)andaC=Ndoublebond(~1.25).

Aconsequenceofthispartialdoublebondcharacteristhat,asfornormaldoublebondstructures,rotationdoesnotoccuraboutthecarbonylcarbontonitrogenofa
peptidebondatphysiologicaltemperatures.Also,aconsequenceoftheC =Ndoublebond'schemistryisthattheatomsattachedtoC andN

alllieinacommonplane.Thusapolypeptidechainisapolymerofpeptidebondplanesinterconnectedatthea carbonatoms.Thea carboninterconnectspeptide

bondsthroughsinglebondsthatallowrotationofadjacentpeptideplaneswithrespecttoeachother.Eachaminoacidresiduecontributesonea carbon(twosingle
bondsandapeptidebond,Figure2.10)tothepolypeptidechain.Thetermresiduereferstotheatomscontributedbyanaminoacidtoapolypeptidechainincluding
theatomsofthesidechain.

ThepeptidebondinFigure2.11ashowsatransconfigurationbetweentheoxygen(O)andthehydrogen(H)atomsofthepeptidebond.Thisisthemoststable
configurationforthepeptidebondwiththetwosidechains(RandR )alsointrans.Thecisconfiguration(Figure2.11b)bringsthetwosidechaingroupstothe
samesideoftheC =Nbond,whereunfavorablerepulsivestericforcesoccurbetweenthetwosidechain(R)groups.Accordingly,transpeptidebondsarealways
foundinproteinsexceptwherethereareprolineresidues.Inprolinethesidechainislinkedtoitsa aminogroup,andthecisandtranspeptidebondswiththe
prolinea iminogrouphavenearequalenergies.Theconfigurationofthepeptidebondactuallyfoundforaprolineinaproteinwilldependonthespecificforces
generatedbytheuniquefoldedthreedimensionalstructureoftheproteinmolecule.

OneofthelargestnaturalpolypeptidechainsinhumansisthatofapolipoproteinB100,whichcontains4536aminoacidresiduesinonepolypeptidechain.Chain
lengthalone,however,doesnotdeterminethefunctionofapolypeptide.Manysmallpeptideswithlessthantenaminoacidsperformimportantbiochemicaland
physiologicalfunctionsinhumans(Table2.2).PrimarystructuresarewritteninastandardconventionandsequentiallynumberedfromtheirNH2terminalendtoward
theirCOOHterminalend,consistentwiththeorderofadditionoftheaminoacidtothechainduringbiosynthesis.Accordingly,forthyrotropinreleasinghormone
(Table2.2)theglutamicacidresiduewrittenontheleftistheNH2terminalaminoacidofthetripeptideandisdesignatedaminoacidresidue1inthesequence.The
prolineistheCOOHterminalaminoacidandisdesignatedresidue3.ThedefineddirectionofthepolypeptidechainisfromGlutoPro(NH2terminalaminoacidto
COOHterminalaminoacid).

Figure2.10
Aminoacidresidue.
Eachaminoacidresidueofapolypeptide
contributestwosinglebondsandonepeptide
bondtothechain.Thesinglebondsarethose
betweentheC andcarbonylCatoms,andthe
a

CaandNatoms.Seep.43fordefinitionoffandy.

Figure2.11
(a)Transpeptidebondand
(b)therare
cispeptidebond.
TheCNhaveapartial
doublebondcharacter.

 Page30

TABLE2.2SomeExamplesofBiologicallyActivePeptides
AminoAcidSequence Name Function
Thyrotropinreleasing Secretedbyhypothalamuscausesanteriorpituitary
hormone glandtoreleasethyrotropichormone

Vasopressin(antidiuretic Secretedbyposteriorpituitaryglandcauseskidneyto
hormone) retainwaterfromurine

Methionineenkephalin Opiatelikepeptidefoundinbrainthatinhibitssenseof
pain

Littlegastrin(human) Hormonesecretedbymucosalcellsinstomachcauses
parietalcellsofstomachtosecreteacid

Glucagon(bovine) Pancreatichormoneinvolvedinregulatingglucose
metabolism

AngiotensinII(horse) Pressororhypertensivepeptidealsostimulates
releaseofaldosteronefromadrenalcortex

Plasmabradykinin(bovine) Vasodilatorpeptide

SubstanceP Neurotransmitter

aTheNH
2terminalGluisinthepyroforminwhichits COOHiscovalentlyjoinedtoits NH2viaamidelinkagetheCOOHterminal
aminoacidisamidatedandthusalsonotfree.
bCysteine1andcysteine6arejoinedtoformadisulfidebondstructurewithinthenonapeptide.

cTheTyr12issulfonatedonitsphenolicsidechainOH.

Figure2.12
Cystinebondformation.

CystineIsaDerivedAminoAcid

Aderivedaminoacidfoundinmanyproteinsiscystine.Itisformedbytheoxidationoftwocysteinethiolsidechains,joinedtoformadisulfidecovalentbond(Figure
2.12).Withinproteinsdisulfidelinksofcystineformedfromcysteines,separatedfromeachotherintheprimarystructure,haveanimportantroleinstabilizingthe
foldedconformationofproteins.

2.3
ChargeandChemicalPropertiesofAminoAcidsandProteins

IonizableGroupsofAminoAcidsandProteinsAreCriticalforBiologicalFunction

IonizablegroupscommontoproteinsandaminoacidsareshowninTable2.3.Theacidformsareontheleftoftheequilibriumsignandthebaseformsontheright
side.Informingitsconjugatebase,theacidformreleasesaproton.Inreverse,thebaseformassociateswithaprotontoformtherespectiveacid.Theproton
dissociationofanacidischaracterizedbyanaciddissociationconstant dependsontheenvironmentinwhichanacidgroupisplaced.Forexample,whena

 Page31

TABLE2.3Characteristic ValuesfortheCommonAcidGroupsinProteins
Approximate
pKaRangefor
WhereAcidGroupIsFound AcidForm BaseForm Group

NH2terminalresidueinpeptides,lysine RNH3
RNH2+H+ 7.610.6
+
Amine
Ammonium

COOHterminalresidueinpeptides,glutamate, RCOOH RCOO+H+ 3.05.5

aspartate Carboxylicacid Carboxylate
Arginine 11.512.5

Cysteine RSH RS+H+ 8.09.0

Thiol Thiolate
Histidine 6.07.0

Tyrosine 9.510.5

positivechargedammoniumgroup(NH3+)isplacednearanegativelychargedgroupwithinaprotein,thenegativechargestabilizesthepositivelychargedacidform
oftheaminogroup,makingitmoredifficulttodissociateitsproton.The valuesandarecalledacidicaminoacids.Theyarepredominantlyintheirunprotonated
formsandarenegativelychargedatphysiologicalpH.Proteinsinwhichtheratio( Lys+ Arg)/( Glu+ Asp)isgreaterthan1arereferredtoasbasicproteins.
Proteinsinwhichtheaboveratioislessthan1arereferredtoasacidicproteins.

TABLE2.4 ofSideChainandTerminalAcidGroupsin
ProteinRibonuclease

NH3+ COOH

Sidechain Lysines 4.6

Chainend Nterminal=7.8 Cterminal=3.8

 Page32

IonicFormofanAminoAcidorProteinCanBeDeterminedataGivenpH

Figure2.13
HendersonHasselbalchequation.
Foramoredetaileddiscussion
ofthisequation,seep.9.

Fromaknowledgeofthe andtheratioof[imidazole]/[imidazolium]is10:1(Table2.5).Basedonthisratio,theenzymeexhibits10/(10+1)100=91%ofits
maximumpotentialactivity.ThusachangeinpHhasadramaticeffectontheenzyme'sactivity.MostproteinactivitiesdemonstratesimilarpHdependencyduetotheir
acidandbasegroup(s).

TitrationofaMonoaminoMonocarboxylicAcid:
DeterminationoftheIsoelectricpH

Anunderstandingofaprotein'sacidandbaseformsandtheirrelationtochargeismademoreclearbyfollowingthetitrationoftheionizablegroupsforasimpleamino
acid.AspresentedinFigure2.14,leucinecontainsana COOHwith .AtpH1.0thepredominantionicform(formI)hasachargeof+1andmigrates
towardthecathodeinan

TABLE2.5RelationshipBetweentheDifferenceofpHand
Acid andtheRatiooftheConcentrationsofBasetoIts
ConjugateAcid

pH RatioofConcentrationof
(DifferenceBetweenpHand BasetoConjugateAcid

0 1
1 10
2 100
3 1000
1 0.1
2 0.01
3 0.001

Figure2.14
Ionicformsofleucine.

 Page33

Figure2.15
Titrationcurveofleucine.

electricalfield.Theadditionof0.5equivalentofbasehalftitratesthea COOHgroupoftheleucinethatis,theratioof[COO]/[COOH]willequal1.The
HendersonHasselbalchequation,withthesecondtermontherightsideoftheequationlog10[(base)/(acid)]=log10[1]=0ataratioofconjugatebasetoacidof1:1,
showsthatthepH(whenthea COOHishalftitrated)isdirectlyequaltothepKa(aCOOH)(Figure2.15).

Additionof1equivalentofbasecompletelytitratesthea COOHbutleavesthea NH3+groupintact.Intheresultingform(II),thenegativelychargeda COOand

positivelychargeda NH3+canceleachotherandthenetchargeofthisionicformiszero.FormIIisthusthezwitterionform,thatis,theionicforminwhichthetotal
ofpositivechargesisexactlyequaltothetotalofthenegativecharges.Asthenetchargeonazwitterionmoleculeiszero,itwillnotmigratetowardeitherthecathode
oranodeinanelectricfield.Furtheradditionof0.5equivalentofbasetothezwitterionformofleucine(totalbaseaddedis1.5equivalents)willthenhalftitratethea
NH3+group.Atthispointinthetitration,theratioof[NH2]/[NH3+]=1,andthepHisequaltothevalueofthe forthea NH3+group(Figure2.15).Additionofa
further0.5equivalentofbase(totalof2fullequivalentsofbaseaddedFigure2.15)completelytitratesthea NH3+grouptoitsbaseform(a NH2).ThesolutionpH
isgreaterthan11,andthepredominantmolecularspecieshasanegativechargeof1(formIII).

ItisusefultocalculatetheexactpHatwhichanaminoacidiselectricallyneutralandinitszwitterionform.ThispHisknownastheisoelectricpHforthemolecule,
andthesymbolispI.ThepIvalueisaconstantofacompoundataparticularionicstrengthandtemperature.Forsimplemolecules,suchasleucine,pIisdirectly
calculatedastheaverageofthetwo valuesthatregulatetheboundariesofthezwitterionform.Leucinehastwoionizablegroupsthatregulatethezwitterionform
boundaries,andthepIiscalculatedasfollows:

AtpH>6.0,leucineassumesapartialnegativechargethatformallyrisesathighpHtoafullnegativechargeof1(formIII)(Figure2.14).AtpH<6,leucinehasa
partialpositivechargeuntilatverylowpHithasachargeof+1(formI)(Figure2.14).ThepartialchargeatanypHcanbecalculatedfromtheHenderson
HasselbalchequationorfromextrapolationfromthetitrationcurveofFigure2.15.

TitrationofaMonoaminoDicarboxylicAcid

AmorecomplicatedexampleoftherelationshipbetweenmolecularchargeandpHisprovidedbyglutamicacid.Itsionizedformsandtitrationcurveare

 Page34

Figure2.16
Ionicformsofglutamicacid.

Figure2.17
Titrationcurveofglutamicacid.

showninFigures2.16and2.17.Inglutamicacidthea COOH valuesthatcontroltheboundariesofthezwitterionform:

Accordingly,atvaluesabovepH3.25themoleculeassumesanetnegativechargeuntilathighpHthemoleculehasanetchargeof2.AtpH<3.25glutamicacidis
positivelycharged,andatextremelylowpHithasanetpositivechargeof+1.

GeneralRelationshipbetweenChargeProperties
ofAminoAcidsandProteinsandpH

AnalysisofchargeformspresentinothercommonaminoacidsshowsthattherelationshipobservedbetweenpHandchargeforleucineandglutamateisgenerallytrue.
Thatis,atasolutionpHlessthanpI,theaminoacidispositivelycharged.AtasolutionpHgreaterthanpI,theaminoacidisnegativelycharged.Thedegreeofpositive
ornegativechargeisafunctionofthemagnitudeofthedifferencebetweenpHandpI.Asaproteinisacomplexpolyelectrolytecontainingmultipleionizableacid
groupsthatregulatetheboundariesofitszwitterionform,calculationofaprotein'sisoelectricpHfromitsacid valuesutilizingtheHendersonHasselbalch
relationshipwouldbedifficult.Accordingly,thepIvaluesforproteinsarealwaysexperimentallymeasuredbydeterminingthepHvalueatwhichtheproteindoesnot
moveinanelectricalfield.pIvaluesforsomerepresentativeproteinsaregiveninTable2.6.

TABLE2.6pIValuesforSome
RepresentativeProteins
Protein pI
Pepsin ~1
Humanserumalbumin 5.9
a1Lipoprotein 5.5

Fibrinogen 5.8
HemoglobinA 7.1
Ribonuclease 7.8
Cytochromec 10.0
Thymohistone 10.6

 Page35

Figure2.18
Relationshipbetweensolution
pH,proteinpI,andproteincharge.

Aswiththeaminoacids,atapHgreaterthanthepI,aproteinhasanetnegativecharge.AtapHlessthanthepI,aproteinhasanetpositivecharge(Figure2.18).
ThemagnitudeofthenetchargeofaproteinincreasesasthedifferencebetweenpHandp/increases.Anexampleishumanplasmaalbuminwith585aminoacid
residuesofwhichthereare61glutamates,36aspartates,57lysines,24arginines,and16histidines.ThetitrationcurveforthiscomplexmoleculeisshowninFigure
2.19.Albumin'spI=5.9,atwhichpHitsnetchargeiszero.AtpH7.5theimidazoliumgroupsofhistidineshavebeenpartiallytitratedandalbuminhasanegative
chargeof10.AtpH8.6additionalgroupshavebeentitratedtotheirbaseforms,andthenetchargeisapproximately20.AtpH11thenetchargeisapproximately
60.OntheacidsideofthepIvalue,atpH3,theapproximatenetchargeofalbuminis+60.

AminoAcidsandProteinsCanBeSeparatedBasedonpIValues

Thetechniquesofelectrophoresis,isoelectricfocusing,andionexchangechromatographyseparateandcharacterizebiologicalmoleculesonthebasisofdifferencesin
theirpI(seep.34).Inclinicalmedicine,separationofplasmaproteinsbyelectrophoresishasledtotheclassificationoftheproteinsbasedontheirrelative
electrophoreticmobility.TheseparationiscommonlycarriedoutatpH8.6,whichishigherthanthepIvaluesofthemajorplasmaproteins.

Figure2.19
Titrationcurveofhumanserumalbuminat25Candan
ionicstrengthof0.150.
RedrawnfromTanford,C.J.Am.Chem.Soc.72:441,1950.

 Page36

Figure2.20
ElectrophoresispatternforplasmaproteinsatpH8.6.
Plotshowstheorderofmigrationalongthehorizontalaxiswithproteinsofhighestmobilityclosest
totheanode.Heightofthebandalongtheverticalaxisshowstheproteinconcentration.Different
majorproteinsaredesignatedunderneaththeirelectrophoreticmobilitypeaks.
ReprintedwithpermissionfromHeide,K.,Haupt,H.,andSchwick,H.G.In:F.W.Putnam(Ed.),
ThePlasmaProteins,2nded.,Vol.III.NewYorkAcademicPress,1977,p.545.

Accordingly,theproteinsarenegativelychargedandmovetowardtheanodeataratedependentontheirnetcharge.Majorpeaksobservedinorderoftheirmigration
arethoseofalbumin,a 1,a 2,andb globulins,fibrinogen,andtheg1andg2globulins(Figure2.20).Someofthesepeaksrepresenttenstohundredsofdifferent
plasmaproteinsthathaveasimilarmigrationrateatpH8.6.However,certainproteinspredominateineachpeakandvariationintheirrelativeamountsischaracteristic
ofcertaindiseases(Figures2.20and2.21seeClin.Corr.2.1).

 Page37

Figure2.21
Examplesoftheelectrophoreticmobilitypatternsobserved
foranormalindividualandpatientswithabnormalconcent
rationsofserumproteins,analyzedbyagarosegel
electrophoresis.
RedrawnfromMcPherson,R.A.Specificproteins.In:J.B.
Henry(Ed.),ClinicalDiagnosisandManagement,17thed.
Philadelphia:SaundersCo,1984.

CLINICALCORRELATION2.1

PlasmaProteinsinDiagnosisofDisease

Electrophoreticanalysisoftheplasmaproteinsiscommonlyusedindiagnosisofdisease.
ElectrophoresisofplasmabufferedatpH8.6separatesthemajorplasmaproteinsasthey
migratetotheanodeintheelectricfieldintobandsorpeaks,basedontheircharge
differences(seetext).ExamplesofabnormalelectrophoresispatternsareshowninFigure
2.21.An''immediateresponse"thatoccurswithstressorinflammationcausedby
infection,injury,orsurgicaltraumaisshowninpattern(b)inwhichhaptoglobinsinthea 2
mobilityhandareselectivelyincreased.A"lateresponse"showninpattern(c)is
correlatedwithinfectionandshowsanincreaseinthetglobulinpeaksduetoanincrease
inimmunoglobulins.Anexampleofahypogammaglobulinemiaduetoan
immunosuppressivediseaseisshowninpattern(d).Inhepaticcirrhosisthereisabroad
elevationofthetglobulinswithreductionofalbumin,asinpattern(e).Monoclonal
gammopathiesareduetotheclonalsynthesisofauniqueimmunoglobulinandgiveriseto
asharptglobulinband,asinpattern(f).Nephroticsyndromeshowsaselectivelossof
lowermolecularweightproteinsfromplasma,asinpattern(g).Thepatternshowsa
decreaseinalbumin(65kDa),butaretentionofthebandscomposedofthehigher
molecularweightproteinsa 2macroglobulin(725kDa)andb lipoproteins(2000kDa)in
thea 2band.Pattern(h)isfromapatientwithaproteinlosingenteropathy.Theslight
increaseinthea 2bandinpattern(h)isduetoanimmediateorlateresponsefromastressful
stimulus,aspreviouslyobservedinpatterns(b)and(c).

Ritzmann,S.E.,andDaniels,J.C.Serumproteinelectrophoresisandtotalserum
proteins.In:S.E.RitzmannandJ.C.Daniels(Eds.),SerumProteinAbnormalities,
DiagnosticandClinicalAspects.Boston:Little,BrownandCo.,1975,pp.325and
McPherson,R.A.Specificproteins.In:J.B.Henry(Ed.),ClinicalDiagnosisand
ManagementbyLaboratoryMethods,17thed.Philadelphia:Saunders,1984,pp.
204215.

 Page38

AminoAcidSideChainsHavePolarorApolarProperties

Therelativehydrophobicityofaminoacidsidechainsiscriticalforthefoldingofaproteintoitsnativestructureandforthestabilityofthefoldedprotein.Figure2.22
plotsthevaluesofrelativehydrophobicityofthecommonaminoacidsbasedonthetendencyofeachaminoacidtopartitionitselfinamixtureofwaterandanonpolar
solvent.Thescaleisbasedonavalueofzeroforglycine.Thesidechainsthatpreferentiallydissolveinthenonpolarsolventrelativetoglycineshowapositive(+)
hydrophobicityvalue,themorepositivethegreaterthepreferenceforthenonpolarsolvent.Mosthydrophobicarethoseaminoacidsfoundburiedinfoldedprotein
structuresawayfromthewatersolventthatinteractswiththesurfaceofasolubleprotein.However,thegeneralcorrelationisnotperfectduetotheamphotericnature
ofmanyofthehydrophobicaminoacidsthatplacethemorepolarportionsoftheirsidechainstructurenearthesurfacetointeractwiththepolarsolventwateronthe
outside.Inaddition,contrarytoexpectation,notallhydrophobicsidechainsareinaburiedpositioninafoldedthreedimensionalstructureofaglobularprotein.When
onthesurface,thehydrophobicgroupsaregenerallydispersedamongthepolarsidechains.Whenclusteringofnonpolarsidechainsoccursonthesurface,itisusually
associatedwithafunctionoftheprotein,suchastoprovideasiteforbindingofsubstratemoleculesthroughhydrophobicinteractions.

Mostchargedsidechainsarefoundonthesurfaceofsolubleglobularproteinswheretheyarestabilizedbyfavorableenergeticinteractionswiththewatersolvent.The
rarepositioningofachargedsidechainintheinteriorofaglobularproteinusuallyimpliesanimportantfunctionalroleforthat"buried"chargewithinthenonpolar
interiorinstabilizingconformationofthefoldedproteinorparticipationinacatalyticmechanism.

AminoAcidsUndergoaVarietyofChemicalReactions

Aminoacidsinproteinsundergoavarietyofchemicalreactionswithreagentsthatmaybeusedtoinvestigatethefunctionofspecificsidechains.Somecommon
chemicalreactionsarepresentedinTable2.7.Reagentsforaminoacidsidechainmodificationhavealsobeensynthesizedthatbindtospecificsitesinafolded
protein'sstructure,likethesubstratebindingsite.Thestrategy

Figure2.22
Relativehydrophobicityoftheaminoacidsidechains.
Basedonthepartitionoftheaminoacidbetweenorganic
solventandwater.Negativevaluesindicatepreferencefor
waterandpositivevaluespreferencefornonpolarsolvent
(ethanolordioxane)relativetoglycine(seetext).
BasedondatafromVonHeijne,G.,andBlomberg,
C.Eur.J.Biochem.97:175,1979andfromNozaki,Y.,
andTanford,C.J.Biol.Chem.246:2211,1971.

 Page39

TABLE2.7SomeChemicalReactionsoftheAminoAcids
ReactiveGroup ReagentorReaction Product

Amine(NH2)groups Ninhydrin Bluecoloredproductthat

absorbsat540nma

Fluorescamine Productthatfluoresces

Carboxylicacidgroups Alcohols Esterproducts

Amines Amideproducts

Carbodiimide Activatesforreactionwith
nucleophiles

NH2ofLys 2,3,6Trinitrobenzene Productthatabsorbsat367nm

sulfonate

Anhydrides Acetylatesamines

Aldehydes FormsShiffbaseadducts

GuanidinogroupofArg Sakaguchireaction Pinkredproductthatcanbeused

toassayArg

PhenolofTyr I2 Iodinationofpositionsorthoto
hydroxylgrouponaromaticring

Aceticanhydride AcetylationofOH

SatomofMetsidechain CH3I Methylsulfoniumproduct

[O]orH2O2 Methioninesulfoxideor
methioninesulfone

SHofCys Iodoacetate Carboxymethylthiolether

product

NEthylmaleimide AdditionproductwithS

Organicmercurials Mercurialadducts

Performicacid Cysteicacid(SO3H)product

Dithionitrobenzoicacid Yellowproductthatcanbeused
toquantitateSHgroups

ImidazoleofHisandphenolof Pauly'sreagent Yellowtoreddishproduct

Tyr
aProlineiminogroupreactswithninhydrintoformproductthatabsorbslightat440nm(yellow

color).

istomodelthestructuralfeaturesoftheenzyme'snaturalsubstrateintothemodifyingreagent.Thereagentbindstotheactivesitelikeanaturalsubstrateand,while
withintheactivesite,reactswithaspecificsidechainintheenzymeactivesite.Thisidentifiesthemodifiedaminoacidasbeinglocatedinthesubstratebindingsiteand
helpsidentifyitsroleinthecatalyticmechanism.

2.4
PrimaryStructureofProteins

Theprimarystructure(aminoacidsequence)ofaproteinisrequiredforanunderstandingofaprotein'sstructure,itsmechanismofactionatamolecularlevel,andits
relationshiptootherproteinswithsimilarphysiologicalroles.Theprimarystructureofinsulinillustratesthevalueofthisknowledgeforunderstandingaprotein's
biosynthesisandphysiologicalforms.Insulinisproducedinpancreaticisletcellsasasinglechainprecursor,proinsulin,withtheprimarystructureshowninFigure
2.23.Thepolypeptidechaincontains86aminoacidsand3intrachaincystinedisulfidebonds.Itistransformedintobiologicallyactiveinsulinbyproteolytic
modificationsinitsprimarystructureasitissecretedfromtheisletcells.Proinsuliniscleavedbyproteasespresentintheisletcellsthatcleavetwopeptidebondsin
proinsulinbetweenresidues30and31and65and66.Thisreleasesa35aminoacidsegment(theCpeptide)

 Page40

Figure2.23
Primarystructuresofhumanproinsulin,insulin,andCpeptide.
Inproinsulin,theBchainpeptideextendsfromPheatposition1toThrat
position30,theCpeptidefromArgatposition31toArgatposition65,andtheAchain
peptidefromGlyatposition66toAsnatposition86.Cystinebondsfrompositions
7to72,19to85,and71to76arefoundinproinsulin.
RedrawnfromBell,G.I.,Swain,W.F.,Pictet,R.,Cordell,B.,
Goodman,H.M.,andRutter,W.J.Nature282:525,1979.

andtheactiveinsulinmolecule.Theactiveinsulinconsistsoftwopolypeptidechains(AandB)of21aminoacidsand30aminoacids,respectively,covalentlyjoined
bythesamedisulfidebondspresentinproinsulin(Figure2.23).TheC

 Page41

TABLE2.8VariationinPositionsA8,A9,A10,andB30of
Insulin
Species A8 A9 A10 B30
Human Thr Ser Ile Thr
Cow Ala Ser Val Ala
Pig Thr Ser Ile Ala
Sheep Ala Gly Val Ala
Horse Thr Gly Ile Ala
Dog Thr Ser Ile Ala
Chickena His Asn Thr Ala

Ducka Glu Asn Pro Thr

aPositions1and2ofBchainarebothAlainchickenand

duckwhereasintheotherspeciesinthetable,position1is
Pheandposition2isValinBchain.

peptideisfurtherprocessedinthepancreaticisletcellsbyproteasesthathydrolyzeadipeptidefromtheCOOHterminalandaseconddipeptidefromtheNH2
terminaloftheCpeptide.ThemodifiedCpeptideissecretedintothebloodwiththeactiveinsulin.Besidesgivinginformationonthepathwayforformationofactive
insulin,knowledgeofprimarystructuresshowstheroleofparticularaminoacidsinthestructureofinsulinthroughcomparisonoftheprimarystructuresoftheinsulins
fromdifferentanimalspecies.Thealignedprimarystructuresshowaresidueidentityinmostaminoacidpositions,exceptforresidues8,9,and10oftheAchainand
residue30oftheBchain.Aminoacidsinthesepositionsvarywidelyindifferentanimalinsulins(Table2.8)andapparentlydonotaffectthebiologicalpropertiesofthe
insulinmolecule(seeClin.Corr.2.2).Otheraminoacidsoftheprimarystructurearerarelysubstituted,suggestingthattheyhaveanessentialroleininsulinfunction.

Comparisonofprimarystructuresiscommonlyusedtopredictthesimilarityinstructureandfunctionbetweenproteins.Sequencecomparisonstypicallyrequire
aligningsequencestomaximizethenumberofidenticalresidueswhileminimizingthenumberofinsertionsordeletionsrequiredtoachievethisalignment.Two
sequencesaretermedhomologouswhentheirsequencesarehighlyalignable.Initscorrectusagehomologyonlyreferstoproteinsthathaveevolvedfromthesame
gene.Analogyisusedtodescribesequencesfromproteinsthatarestructurallysimilarbutforwhichnoevolutionaryrelationshiphasbeendemonstrated.Substitution
ofanaminoacidbyanotheraminoacidofsimilar

CLINICALCORRELATION2.2
DifferencesinPrimaryStructureofInsulinsUsedinTreatmentofDiabetes
Mellitus

Bothpig(porcine)andcow(bovine)insulinsarecommonlyusedinthetreatmentof
humandiabetics.Becauseofthedifferencesinaminoacidsequencefromthehuman
insulin,somediabeticindividualswillhaveaninitialallergicresponsetotheinjectedinsulin
astheirimmunologicalsystemrecognizestheinsulinasforeign,ordevelopaninsulin
resistanceduetoahighantiinsulinantibodytiteratalaterstageintreatment.However,
thenumberofdiabeticswhohaveadeleteriousimmunologicalresponsetopigandcow
insulinsissmallthegreatmajorityofhumandiabeticscanutilizethenonhumaninsulins
withoutimmunologicalcomplication.Thecompatibilityofcowandpiginsulinsinhumans
isduetothesmallnumberandtheconservativenatureofthechangesbetweentheamino
acidsequencesoftheinsulins.Thesechangesdonotsignificantlyperturbthethree
dimensionalstructureoftheinsulinsfromthatofhumaninsulin.Piginsulinisusuallymore
acceptablethancowinsulinininsulinreactiveindividualsbecauseitismoresimilarin
sequencetohumaninsulin(seeTable2.8).Humaninsulinisnowavailableforclinicaluse.
Itcanbemadeusinggeneticallyengineeredbacteriaorbymodifyingpiginsulin.

Brogdon,R.N.,andHeel,R.C.Humaninsulin:areviewofitsbiologicalactivity,
pharmacokinetics,andtherapeuticuse.Drugs34:350,1987.

 Page42

CLINICALCORRELATION2.3

ANonconservativeMutationOccursinSickleCellAnemia

HemoglobinS(HbS)isavariantformofthenormaladulthemoglobininwhicha
nonconservativesubstitutionoccursinthesixthpositionoftheb polypeptidechainofthe
normalhemoglobin(HbA1).WhereasinHbA1thispositionistakenbyaglutamicacid
residue,inHbSthepositionisoccupiedbyavaline.Consequently,inHbSapolarside
chaingrouponthemolecule'soutsidesurfacehasbeenreplacedwithanonpolar
hydrophobicsidechain(anonconservativemutation).Throughhydrophobicinteractions
withthisnonpolarvaline,HbSinitsdeoxyconformationpolymerizeswithothermolecules
ofdeoxyHbS,leadingtoaprecipitationofthehemoglobinwithintheredbloodcell.This
precipitationmakestheredbloodcellassumeasickleshapethatresultsinahighrateof
hemolysisandalackofelasticityduringcirculationthroughthesmallcapillaries,which
becomecloggedbytheabnormalshapedcells.

OnlyindividualshomozygousforHbSexhibitthedisease.Individualsheterozygousfor
HbShaveapproximately50%HbA1and50%HbSintheirredbloodcellsanddonot
exhibitsymptomsofthesicklecellanemiadiseaseexceptunderextremeconditionsof
hypoxia.

IndividualsheterozygousforHbShavearesistancetothemalariaparasite,whichspends
apartofitslifecycleinredbloodcells.ThisisafactorselectingfortheHbSgenein
malarialregionsoftheworldandisthereasonforthehighfrequencyofthislethalgenein
thehumangeneticpool.Approximately10%ofAmericanblacksareheterozygousfor
HbS,and0.4%ofAmericanblacksarehomozygousforHbSandexhibitsicklecell
anemia.

HbSisdetectedbygelelectrophoresis.Becauseitlacksaglutamate,itislessacidicthan
HbA.HbSthereforedoesnotmigrateasrapidlytowardtheanodeasdoesHbA.Itis
alsopossibletodiagnosesicklecellanemiabyrecombinantDNAtechniques.

Embury,S.H.Theclinicalpathophysiologyofsicklecelldisease.Annu.Rev.Med.
37:361,1986.

polarity(i.e.,ValforIleinposition10ofinsulin)iscalledaconservativesubstitutionandiscommonlyobservedinaminoacidsequencesofthesameproteinfrom
differentanimalspecies.Ifaparticularaminoacidisalwaysfoundatthesamepositioninthesecomparisons,thenthesearedesignatedinvariantresiduesanditcan
beassumedthattheseresidueshaveanessentialroleinthestructureorfunctionoftheprotein.Incontrast,anonconservativesubstitutioninvolvesreplacementofan
aminoacidbyanotherofdramaticallydifferentpolarity.Thismayproduceseverechangesinthepropertiesoftheresultantproteinoroccurinregionsthatare
apparentlyunimportantfunctionally(seeClin.Corr.2.3).Polarityisonlyonephysicalpropertyofaminoacidsthatdetermineswhetherasubstitutionwillsignificantly
altertheprotein'sfunction.Otherphysicalpropertiesofimportancearethevolumeandsurfacearea.

2.5
HigherLevelsofProteinOrganization

Primarystructureofaproteinreferstothecovalentstructureofaprotein.Itincludesaminoacidsequenceandlocationofdisulfide(cystine)bonds.Higherlevelsof
proteinorganizationrefertononcovalentlygeneratedconformationalpropertiesoftheprimarystructure.Thesehigherlevelsofproteinconformationandorganization
aredefinedasthesecondary,tertiary,andquaternarystructuresofaprotein.Secondarystructurereferstothelocalthreedimensionalfoldingofthepolypeptide
chainintheprotein.Thepolypeptidechaininthiscontextisthecovalentlyinterconnectedatomsofthepeptidebondsanda carbonlinkagesthatsequentiallylinkthe
aminoacidresiduesoftheprotein.Sidechainsarenotconsideredatthelevelofsecondarystructure.Tertiarystructurereferstothethreedimensionalstructureof
thepolypeptide.Itincludestheconformationalrelationshipsinspaceofthesidechainsandthegeometricrelationshipbetweendistantregionsofthepolypeptidechain.
Quaternarystructurereferstothestructureandinteractionsofthenoncovalentassociationofdiscretepolypeptidesubunitsintoamultisubunitprotein.Notall
proteinshaveaquaternarystructure.

Proteinsgenerallyassumeuniquesecondary,tertiary,andquaternaryconformationsasdeterminedbytheirparticularaminoacidsequenceandtermedthenative
conformation.Foldingoftheprimarystructureintothenative

 Page43

conformationoccurs,inmostcases,spontaneouslythroughnoncovalentinteractions.ThisuniqueconformationistheoneoflowesttotalGibbsfreeenergykinetically
accessibletothepolypeptidechain(s)fortheparticularconditionsofionicstrength,pH,andtemperatureofthesolventinwhichthefoldingoccurs.Chaperoneproteins
mayfacilitatetherateofproteinfolding.

Figure2.24
Polypeptidechainshowingf,y,andpeptidebonds
forresidueRiwithinapolypeptidechain.
RedrawnwithpermissionfromDickerson,R.E.,
andGeis,I.TheStructureandActionofProteins.
MenloPark,CA:Benjamin,1969,p.25.

ProteinsHaveaSecondaryStructure

Theconformationofapolypeptidechainmaybedescribedbytherotationalanglesofthecovalentbondsthatcontributetothepolypeptidechain.Thesearethebonds
contributedbyeachoftheaminoacidsbetween(1)thenitrogenanda carbonand(2)thea carbonandthecarbonylcarbon.Thefirstoftheseisdesignatedthephi
(f )bondandthesecondiscalledthepsi(y )bondforanaminoacidresidueinapolypeptidechain(Figure2.24).Thethirdbondcontributedbyeachaminoacidto
thepolypeptidechainisthepeptidebond.Aspreviouslydiscussed,duetothepartialdoublebondcharacterofthe bonds,thereisabarriertofreerotation
aboutthispeptidebond.

Regularsecondarystructureconformationsinsegmentsofapolypeptidechainoccurwhenallf bondanglesinthatpolypeptidesegmentareequal,andallthey
bondanglesareequal.Therotationalanglesforf andy bondsforcommonregularsecondarystructuresaregiveninTable2.9.

Thea helixandb structureconformationsforpolypeptidechainsarethemostthermodynamicallystableoftheregularsecondarystructures.However,aparticular

sequencemayformregularconformationsotherthana helicalorb structure.Therearealsoregionsofunorderedsecondarystructure,inwhichneitherthef bond
anglesnorthey bondanglesareequal.Prolineinterruptsa helicalconformationssincethepyrrolidinesidechainofprolinestericallyinteractswiththeaminoacid
precedingitinthepolypeptidesequencewheninana helicalstructure.Thisrepulsivestericinteractiontendstopreventformationofa helicalstructureinsectionsofa
polypeptidechainthatcontainproline.

Helicalstructuresofpolypeptidechainsarecharacterizedbythenumberofaminoacidresiduesperturnofhelix(n)andthedistancebetweena carbonatomsof
adjacentaminoacidsmeasuredparalleltotheaxisofthehelix(d).Thehelixpitch(p),definedastheproductofnd,thenmeasuresthedistancebetweenrepeating
turnsofthehelixonalinedrawnparalleltothehelixaxis(Figure2.25):

a HelicalStructure

Anaminoacidsequenceinana helicalconformationisshowninFigure2.26.

Figure2.25
Thehelixpitch(p)fora
helixwithn=4.
Eachcircleonalinerepre
sentsan carbonfroman
aminoacidresidue.Therise
perresiduewouldbe
p/n(seeequationintext).
FromDickerson,R.E.,and
Geis,I.TheStructureand
ActionofProteins.Menlo
Park,CA:Benjamin,1969,p.26.

TABLE2.9HelixParametersofRegularSecondaryStructures
ApproximateBond Helix
Angles()
Residuesper Pitch,ap
Structure f y turn,n (A)

Righthandedahelix[3.613 57 47 3.6 5.4

helix)

310helix +49 26 3.0 6.0

Parallelbstrand 119 +113 2.0 6.4

Antiparallelbstrand 139 +135 2.0 6.8

PolyprolinetypeIIb 78 +149 3.0 9.4

a
Distancebetweenrepeatingturnsonalinedrawnparalleltohelixaxis.
bHelixtypefoundforpolypeptidechainsofcollagen.

 Page44

360turn(n=3.6).Thepeptidebondplanesinthea helixareparalleltotheaxisofthehelix.Inthisgeometryeachpeptideformstwohydrogenbonds,onetothe
peptidebondofthefourthaminoacidaboveandthesecondtothepeptidebondofthefourthaminoacidbelowintheprimarystructure.Othera helixparameters,
suchasthepitch(p),aregiveninTable2.9.Inthehydrogenbondsbetweenthepeptidegroupsofana helicalstructure,thedistancebetweenthehydrogendonor
atomandthehydrogenacceptoratomis2.9.Also,thedonor,acceptor,andhydrogenatomsareapproximatelycollinear,inthattheydetermineastraightline.This
isanoptimumgeometryanddistanceformaximumhydrogenbondstrength(seeSection2.7).

Figure2.26
Anahelix.
Redrawnwithpermission,based
onfigurefromPauling,L.The
NatureoftheChemicalBond,
3rded.Ithaca,NY:Cornell
UniversityPress,1960.

Thesidechainsinana helicalconformationareontheoutsideofthespiralstructuregeneratedbythepolypeptidechain.Duetothecharacteristic3.6residuesper
turn,thefirstandeverythirdandfourthRgroupoftheaminoacidsequenceinthehelixcomeclosetotheother.Helicesoftenpresentseparablepolarandnonpolar
facesbasedontheiraminoacidsequences,whichplacepolarornonpolarsidechainsthreeorfouraminoacidsapartinthesequence,whichfoldsintothea helix.
Thiswillgiverisetouniquefunctionalcharacteristicsofthehelix.However,ifeverythirdorfourthsidechainthatcomeclosetogetherhavethesamechargesignorare
branchedattheirb carbon(valineandisoleucine),theirunfavorableionicorstericinteractionsdestabilizethehelixstructure.Thea helixmaytheoreticallyformits
spiralineitheralefthandedorrighthandedsense,givingthehelixasymmetricpropertiesandcorrelatedopticalactivity.Inthestructureshown,arighthandeda helix
isdepictedthisismorestablethanthelefthandedhelix.

b Structure

Apolypeptidechaininab strandconformation(Figure2.27)ishydrogenbondedtoanothersimilarstrandalignedeitherinaparallelorantiparalleldirection(Figure
2.28).Hydrogenbondedb strandsappearlikeapleatedsheet(Figure2.29).Thesidechainsprojectaboveandbelowthepleatedsheetlikestructure.

SupersecondaryStructures

Certaincombinationsofsecondarystructurecanbeobservedindifferentfoldedproteinstructures.Theyarereferredtoasstructuralmotifsandincludehelixturn
helix(seep.108),leucinezipper(seep.110),calciumbindingEFhand(seep.209),andzincfinger(seep.108).Evenlongerorderingsmayoccurtoformadomain
(seebelow)suchastheb barrelandtheimmunoglobulinfold.Theselongerpatternlengthsofsecondarystructuremayincludemultiplestructuralmotifsandwhen
commonlyobservedinmorethanoneproteinarereferredtoassupersecondarystructures.

ProteinsFoldintoaThreeDimensional
StructureCalledtheTertiaryStructure

Thetertiarystructureofaproteinisthethreedimensionalstructureofaprotein.Itincludesthegeometricrelationshipbetweendistantsegmentsofprimarystructure
andtherelationshipofthesidechainswithoneanotherinthreedimensionalspace.Asanexampleofaprotein'stertiarystructure,thestructurefortrypsinisshownin
Figure2.30.InFigure2.30atheribbonstructureshowstheconformationofpolypeptidestrandsandtheoverallpatternofpolypeptidechainfolding(supersecondary
structure).ThetertiarystructureisthenfurtherbuiltuponinFigure2.30bbyshowingthesidechaingroupsandtheirinterconnectionswithastickmodel.Activesite
catalyticsidechainsareshowninyellow,whichincludethehydroxymethylgroupofserine(residue177inthesequence),theimidazoleofhistidine(residue40),and
thecarboxylatecontainingsidechainofaspartate(residue85).Althoughthesecatalyticresidues

 Page45

Figure2.27
Twopolypeptidechainsinabstructureconformation.
Additionalpolypeptidechainsmaybeaddedtogeneratemoreextendedstructure.
RedrawnwithpermissionfromFersht,A.EnzymeStructureandMechanism,SanFrancisco:
Freeman,1977,p.10.

Figure2.28
Exampleofantiparallelbstructure(residues9398,2833,and1621of
Cu,Znsuperoxidedismutase).
Dashedlineshowshydrogenbondsbetweencarbonyloxygenatomsand
peptidenitrogenatomsarrowsshowdirectionofpolypeptidechainsfromNterminalto
Cterminal.Inthecharacteristicantiparallel structure,pairsofcloselyspacedinterchain
hydrogenbondsalternatewithwidelyspacedhydrogenbondpairs.
RedrawnwithpermissionfromRichardson,J.S.Adv.ProteinChem.34:168,1981.

Figure2.29
bPleatedsheetstructurebetweentwopolypeptidechains.
Additionalpolypeptidechainsmaybeaddedaboveandbelowtogenerate
amoreextendedstructure.

 Page46

Figure2.30
Tertiarystructureoftrypsin.
(a)Ribbonstructureoutlinestheconformationofthepolypeptidechain.
(b)Structureshowssidechainsincludingactivesiteresidues(inyellow)withoutlineofpolypeptidechain(ribbon)superimposed.
(c)SpacefillingstructureinwhicheachatomisdepictedasthesizeofitsvanderWaalsradius.Hydrogenatomsare
notshown.Differentdomainsareshownindarkblueandwhite.Theactivesiteresiduesareinyellowandintrachain
disulfidebondsofcystineinred.Lightbluespheresrepresent
watermoleculesassociatedwiththeprotein.Thisstructureshowsthedensityofpackingwithintheinterioroftheprotein.

arewidelyseparatedintheprimarystructure,thefoldedtertiarystructurebringsthemtogetherinspacetoformthecatalyticsite.InFigure2.30caspacefillingmodel
showsC,N,andOatomsrepresentedbyballsofradiusproportionaltotheirvanderWaalsradius.

Thetertiarystructureoftrypsinconformstothegeneralrulesoffoldedproteins(seeSection2.3).Hydrophobicsidechainsaregenerallyintheinteriorofthestructure,
awayfromthewaterinterface.Ionizedsidechainsoccurontheoutsideofaproteinstructure,wheretheyarestabilizedbywaterofsolvation.Withintheprotein
structure(notshown)areburiedwatermolecules,noncovalentlyassociated,whichexhibitspecificarrangements.Alargenumberofwatermoleculesformasolvation
shellaroundtheoutsideoftheprotein.

Alongpolypeptidestrandoftenfoldsintomultiplecompactsemiindependentfoldedregionsordomains,eachdomainhavingacharacteristiccompactgeometrywith
ahydrophobiccoreandpolaroutside.Theytypicallycontain100150contiguousaminoacids.Thedomainsofamultidomainproteinmaybeconnectedbya
segmentofthepolypeptidechainlackingregularsecondarystructure.Alternatively,thedensesphericalfoldedregionsareseparatedbyacleftorlessdenseregionof
tertiarystructure(Figure2.31).Therearetwofoldeddomainsinthetrypsinmoleculewithacleftbetweenthedomains

 Page47

thatincludesthesubstratebindingcatalyticsiteoftheprotein.Anactivesitewithinaninterdomaininterfaceisanattributeofmanyenzymes.Differentdomainswithina
proteincanmovewithrespecttoeachother.Hexokinase(Figure2.32),whichcatalyzesphosphorylationofaglucosemoleculebyadenosinetriphosphate(ATP),has
theglucosebindingsiteinaregionbetweentwodomains.Whentheglucosebindsintheactivesite,thesurroundingdomainsmovetoenclosethesubstratetotrapit
forphosphorylation(Figure2.32).Inenzymeswithmorethanonesubstrateorallostericeffectorsite(seeChapter4),thedifferentsitesmaybelocatedwithindifferent
domains.Inmultifunctionalproteins,eachdomainperformsadifferenttask.

Figure2.31
Globulardomainswithinproteins.
(a)Phosphoglyceratekinasehas
twodomainswitharelativelynarrowneck
inbetween.
(b)Elastasehastwotightly
associateddomainsseparatedbyanarrow
cleft.Eachsphereinthespacefilling
drawingrepresentsthe carbonpositionforan
aminoacidwithintheproteinstructure.
ReprintedwithpermissionfromRichardson,
J.S.Adv.ProteinChem.34:168,1981.

HomologousThreeDimensionalDomainStructuresAreOftenFormedfromCommonArrangementsofSecondaryStructures

Aproteincanadoptarangeofconformationsforaparticularaminoacidsequence.Althougheachnativestructureisunique,acomparisonofthetertiarystructuresof
differentproteinssolvedbyXraycrystallographyshowssimilararrangementsofsecondarystructuremotifsthatformthetertiarystructuresofdomains.Thusproteins
unrelatedbyfunction,sequence,orevolutionshowsimilarpatternsofarrangementoftheirsecondarystructuresorsupersecondary

Figure2.32
Drawingsof(a)unligandedform
ofhexokinaseandfreeglucoseand
(b)theconformationofhexokinasewithglucose
bound.Inthisspacefillingdrawingeach
circlerepresentsthevanderWaalsradius
ofanatominthestructure.Glucoseis
black,andeachdomainisdifferentlyshaded.
Reprintedwithpermission
fromBennett,W.S.,andHuber,R.CRCRev.
Biochem.15:291,1984.
CopyrightCRCPress,Inc.,BocaRaton,FL.

 Page48

Figure2.33
Anexampleofanallafoldeddomain.
Inthisdrawingandthosethatfollow
(Figures2.342.36),onlythe
outlineofthepolypeptidechainis
shown. Structure
strandsareshownbyarrowswiththe
directionofthearrowshowingthe
N Cterminal
directionofthechainlightningbolts
representdisulfidebonds,andcircles
representmetalioncofactors(whenpresent).
Redrawnwithpermissionfrom
Richardson,J.S.Adv.Protein
Chem.34:168,1981.

structures.Aclassificationsystemforsupersecondarypatternsplacescommonfoldingpatternsforsecondarystructuresintostructuralfamilies.Thekeysuper
secondarystructuresareformedbecauseofthethermodynamicstabilityoftheirfoldingpatterns.

Acommonall a structureisfoundintheenzymelysozyme(Figure2.33).Otherexamplesofall a structureareinmyoglobinandthesubunitsofhemoglobin,whose

structuresarediscussedinChapter3.Inthissupersecondaryfoldingpattern,sevenoreightsectionsofa helicesarejoinedbysmallersegmentsofpolypeptidechains
thatallowthehelicestofoldbackuponthemselvestoformacharacteristicglobularshape.Anothercommonsupersecondarystructureisthea ,b domainstructure
shownbytriosephosphateisomerase(Figure2.34)inwhichthestrands(designatedbyarrows)arewoundintoab barrel.Eachb strandintheinterioroftheb
barrelisinterconnectedbya helicalregionsofthepolypeptidechainontheoutsideofthemolecule.Asimilarsupersecondarystructureisfoundinpyruvatekinase
(Figure2.34).Adifferenttypeofa ,b domainsupersecondarystructureisseeninlactatedehydrogenaseandphosphoglyceratekinase(Figure2.35).Inthesethe
interiorpolypeptidesectionsparticipateinatwistedsheetb structure.Theb structuresegmentsarejoinedbya helixregionspositionedontheoutsideofthe
moleculetogiveacharacteristica ,b domainfoldingpattern.Anallb domainsupersecondarystructureispresentinCu,Znsuperoxidedismutase,inwhichthe
antiparallelb sheetformsaGreekkeyb barrel(Figure2.36).Asimilarpatternoccursineachofthedomainsoftheimmunoglobulins,discussedinChapter3.
ConcanavalinA(Figure2.36)showsanall b domainstructureinwhichtheantiparallelb strandsformab barrelpatterncalleda''jellyroll."Proteinstructuresusedto
definetheseclasseshavebeenobservedbyXraycrystallographicanalysis(Section2.9),primarilyofglobularproteinsthatarewatersoluble.Proteinsthatarenot
watersolublemaycontaindifferentsupersecondarypatterns(seeSection2.6).

Figure2.34
Examplesofa,bfoldeddomainsinwhich
bstructuralstrandsforma
bbarrelinthecenterofthe
domain(seelegendtoFigure2.33).
Redrawnwithpermissionfrom
Richardson,J.S.Adv.Protein
Chem.34:168,1981.

AQuaternaryStructureOccursWhenSeveralPolypeptideChainsFormaSpecificNoncovalentAssociation

Quaternarystructurereferstothearrangementofpolypeptidechainsinamultichainprotein.Thesubunitsinaquaternarystructuremustbeinnoncovalentassociation,
a Chymotrypsincontainsthreepolypeptidechainscovalentlyjoinedtogetherbyinterchaindisulfidebondsintoasinglecovalentunitandthereforedoesnothavea
quaternarystructure.Myoglobinconsistsofasinglepolypeptidechainandhasnoquaternarystructure.However,hemoglobinA

 Page49

Figure2.35
Examplesofa,bfolded
domainsinwhichbstructurestrandsareintheformof
aclassicaltwistedbsheet(seelegendtoFigure2.33).
RedrawnwithpermissionfromRichardson,J.S.Adv.ProteinChem.
34:168,1981.

Figure2.36
Examplesofallbfoldeddomains(seelegendtoFigure2.33).
RedrawnwithpermissionfromRichardson,J.S.Adv.
ProteinChem.34:168,1981.

containsfourpolypeptidechains(a 2b 2)heldtogethernoncovalentlyinaspecificconformationasrequiredforitsfunction(seeChapter3).Thushemoglobinhasa
quaternarystructure.Aspartatetranscarbamylase(seeChapter13)hasaquaternarystructurecomprisedof12polypeptidesubunits.Thepoliovirusproteincoat
contains60polypeptidesubunits,andthetobaccomosaicvirusproteinhas2120polypeptidesubunitsheldtogethernoncovalentlyinaspecificstructuralarrangement.

2.6
OtherTypesofProteins

Thecharacteristicsofproteinstructure,discussedabove,arebasedonobservationsonglobular,watersolubleproteins.Otherproteins,suchasthefibrousproteins,
arenonglobularandhavealowwatersolubilitylipoproteinsand

 Page50

glycoproteinshaveaheterogeneouscompositionandmayormaynotbewatersoluble.

FibrousProteinsIncludeCollagen,Elastin,a Keratin,andTropomyosin

Globularproteinshaveaspheroidalshape,variablemolecularweights,relativelyhighwatersolubility,andavarietyoffunctionalrolesascatalysts,transporters,and
controlproteinsfortheregulationofmetabolicpathwaysandgeneexpression.Incontrast,fibrousproteinscharacteristicallycontainlargeramountsofregular
secondarystructure,alongcylindrical(rodlike)shape,alowsolubilityinwater,andastructuralratherthanadynamicroleinthecellororganism.Examplesoffibrous
proteinsarecollagen,a keratin,andtropomyosin.

DistributionofCollageninHumans

Collagenispresentinalltissuesandorganswhereitprovidestheframeworkthatgivesthetissuestheirformandstructuralstrength.Itsimportanceisshownbyits
highconcentrationinallorgansthepercentageofcollagenbyweightforsomerepresentativehumantissuesandorgansis4%oftheliver,10%oflung,1224%ofthe
aorta,50%ofcartilage,64%ofthecornea,23%ofwholecorticalbone,and74%ofskin(seeClin.Corr.2.4).

AminoAcidCompositionofCollagen

Theaminoacidcompositionofcollagenisquitedifferentfromthatofatypicalglobularprotein.TheaminoacidcompositionoftypeIskincollagenandofglobular
proteinsribonucleaseandhemoglobinaregiveninTable2.10.Skincollageniscomparativelyrichinglycine(33%ofitsaminoacids),proline(13%),thederivedamino
acid4hydroxyproline(9%),andanotherderivedaminoacid5hydroxylysine(0.6%)(Figure2.37).Hydroxyprolineisuniquetocollagensbeingformedenzymatically
fromprolineswithinacollagenpolypeptidechain.Theenzymecatalyzedhydroxylationofprolinerequiresthepresenceofascorbicacid(vitaminC)thusinvitaminC
deficiency(scurvy)thereispoorsynthesisofnewcollagen.Mosthydroxyprolinesinacollagenhavethehydroxylgroupinthe4position(gcarbon)oftheproline
structure,althoughasmallamountof3hydroxyprolineisalsoformed(Table2.10).Collagensareglycoproteinswithcarbohydratecovalentlyjoinedtothederived
aminoacid,5hydroxylysine,byanOglycosidicbondthroughthed carbonhydroxylgroup.Formationof5hydroxylysinefromlysinesandadditionofthe
carbohydratetothe5hydroxylysineoccurafterpolypeptidechainformationbutpriortothefoldingofthecollagenchainsintotheiruniquesupercoiledstructure.

Figure2.37
Derivedaminoacidsfoundincollagen.
Carbohydrateisattachedto5OH
in5hydroxylysinebyatypeIII
glycosidiclinkage(seeFigure2.45).

AminoAcidSequenceofCollagen

Themolecularunitofmaturecollagenortropocollagencontainsthreepolypeptidechains.Variousdistinctcollagenchainsexistthatmakeupthedifferent

CLINICALCORRELATION2.4
SymptomsofDiseasesofAbnormalCollagenSynthesis

Collagenispresentinvirtuallyalltissuesandisthemostabundantproteininthebody.
Certainorgansdependheavilyonnormalcollagenstructuretofunctionphysiologically.
Abnormalcollagensynthesisorstructurecausesdysfunctionofcardiovascularorgans
(aorticandarterialaneurysmsandheartvalvemalfunction),bone(fragilityandeasy
fracturing),skin(poorhealingandunusualdistensibility),joints(hypermobilityand
arthritis),andeyes(dislocationofthelens).Examplesofdiseasescausedbyabnormal
collagensynthesisincludeEhlersDanlossyndrome,osteogenesisimperfecta,andscurvy.
Thesediseasesmayresultfromabnormalcollagengenes,abnormalposttranslational
modificationofcollagen,ordeficiencyofcofactorsneededbytheenzymesthatcarryout
posttranslationalmodificationofcollagen.

Byers,P.H.Disordersofcollagenbiosynthesisandstructure.In:C.R.Scriver,A.L.
Beaudet,W.S.Sly,andD.Valle(eds.),TheMetabolicandMolecularBasesof
InheritedDisease,7thed.McGrawHill,1995,Chap.134.

 Page51

TABLE2.10ComparisonoftheAminoAcidContentofHumanSkinCollagen(TypeI)andMatureElastinwith
ThatofTwoTypicalGlobularProteinsa
Collagen Ribonuclease Hemoglobin
AminoAcid (HumanSkin) Elastin(Mammalian) (Bovine) (Human)
COMMONAMINOACIDS PERCENTOFTOTAL
Ala 11 8 9

Arg 5 0.9 5 3
Asn 8 3

Asp 5 1 15 10
Cys 0 0 0 1
Glu 7 2 12 6
Gln 6 1

Gly 2 4

His 0.5 0.1 4 9
Ile 1 2 3 0
Leu 2 6 2 14
Lys 3 0.8 11 10
Met 0.6 0.2 4 1
Phe 1 3 4 7
Pro 4 5

Ser 4 1 11 4
Thr 2 1 9 5
Trp 2 1 9 2
Tyr 0.3 2 8 3
Val 2 12 8 10
DERIVEDAMINOACIDS
Cystine 0 0 7 0
3Hydroxyproline 0 0

4Hydroxyproline 0

5Hydroxylysine 0 0 0

Desmosineandisodesmosine 0 0 0

a
Boxednumbersemphasizeimportantdifferencesinaminoacidcompositionbetweenthefibrousproteins
(collagenandelastin)andtypicalglobularproteins.

collagentypes,eachwiththeirowngenes.Insometypes,thethreepolypeptidechainshaveanidenticalaminoacidsequence.InotherssuchastypeI(Table2.11),
twoofthechainsareidenticalwhiletheaminoacidsequenceofthethirdchainisslightlydifferent.IntypeIcollagen,theidenticalchainsaredesignateda 1(I)chains
andthethirdnonidenticalchain,a 2(I).IntypeVcollagenallthreechainsaredifferent,designateda 1(V),a 2(V),anda 3(V).Differenttypesofcollagendifferintheir
physicalpropertiesduetodifferencesintheaminoacidsequenceamongchains,eventhoughtherearelargeregionsofhomologoussequenceamongthedifferentchain
types.Collagenhascovalentlyattachedcarbohydrateandthecollagentypesdifferintheircarbohydratecomponent.Table2.11describessomecharacteristicsof
collagentypesIVIadditionalcollagentypes(designatedupthroughtypeXVI)havebeenreported.

Theaminoacidsequenceofthechainsofcollagensisunusual.Inlongsegmentsofallthecollagentypesglycineoccursaseverythirdresidueandprolineor
hydroxyprolinealsooccursthreeresiduesapartinthesesameregions.Accordingly,theaminoacidsequencesGlyProYandGlyXHyp(whereXandYareanyof
theaminoacids)arerepeatedintandemseveralhundredtimes.IntypeIcollagen,thetripletsequencesarereiteratedover200times,encompassingover600amino
acidswithinachainofapproximately1000aminoacids.

 Page52

TABLE2.11ClassificationofCollagenTypes
Chain
Type Designations TissueFound Characteristics
I [a1(I)]2a2(I) Bone,skin,tendon,scar Lowcarbohydrate<10
tissue,heartvalve,intestinal, hydroxylysinesperchaintwo
anduterinewall typesofpolypeptidechains
II [a1(II)]3 Cartilage,vitreous 10%carbohydrate>20
hydroxylysinesperchain
III [a1(III)]3 Bloodvessels,newbornskin, Lowcarbohydratehigh
scartissue,intestinal,and hydroxyprolineandGly
uterinewall containsCys

IV [a1(IV)]3 Basementmembrane,lens High3hydroxyproline>40
capsule hydroxylysinesperchainlow
[a2(IV)]3
AlaandArgcontainsCys
highcarbohydrate(15%)
V [a1(V)]2a2(V)[a1 Cellsurfacesor Highcarbohydrate,relatively
exocytoskeletonwidely highglycine,and
(V)]3a1(V)a2(V)
distributedinlowamounts hydroxylysine
a3(V)
VI Aorticintima,placenta, Relativelylargeglobular
kidney,andskininlow domainsintelopeptide
amounts regionhighCysandTyr
molecularweightrelatively
low(~160,000)equimolar
amountsofhydroxylysine
andhydroxyproline

StructureofCollagen

Polypeptidesthatcontainonlyprolinecanbesynthesizedinthelaboratory.Thesepolyprolinechainsassumearegularsecondarystructureinaqueoussolutioninwhich
thechainisinatightlytwistedextendedhelixwiththreeresiduesperturnofthehelix(n=3).Thishelixwithalltranspeptidebondsisdesignatedthepolyproline
typeIIhelix(seeFigure2.11fordifferencesbetweencisandtranspeptidebonds).Thepolyprolinehelixhasthesamecharacteristicsasthehelixfoundincollagen
chainsinregionsoftheprimarystructurethatcontainaprolineorhydroxyprolineatapproximatelyeverythirdposition.Sincethehelixstructureincollagenisthesame
asthatofpolyproline,thethermodynamicforcesleadingtoformationofthecollagenhelixstructureareduetothepropertiesofproline.Inproline,thef angle
contributedtothepolypeptidechainispartofthefivemembercyclicsidechain.ThefivememberringconstrainstheCaNbondtoananglecompatiblewiththe
polyprolinehelixstructure.

InpolyprolinetypeIIhelix,theplaneofeachpeptidebondisperpendiculartotheaxisofthehelix.Inthisgeometrythepeptidecarbonylgroupsarepointedtoward
neighboringchainsandarecorrectlyorientedtoformstronginterchainhydrogenbondswithotherchainsofthecollagenmolecule.Thisisincontrasttothea helix,in
whichtheplanecontainingtheatomsofthepeptidebondis

 Page53

Figure2.38
Diagramofcollagendemonstratingnecessityforglycineineverythirdresidue
toallowthedifferentchainstobeincloseproximityinthestructure.
(a)Ribbonmodelforsupercoiledstructureofcollagenwitheach
individualchaininapolyprolinetypeIIhelix.
(b)Moredetailedmodelofsupercoiledconformation.
All carbonatomsarenumberedand
proposedhydrogenbondsareshownbydashedlines.
RedrawnwithpermissionfromDickerson,R.E.,andGeis,
I.TheStructureandActionsofProteins,
MenloPark,CA:Benjamin,1969,pp.41,42.

paralleltothea helixaxisandthepeptidebondsformonlyintrachainhydrogenbondswithpeptidebondsinthesamepolypeptidechain.Thethreechainsofa
collagenmolecule,whereeachofthechainsisinapolyprolinetypeIIhelixconformation,arewoundabouteachotherinadefinedwaytoformasuperhelical
structure(Figure2.38).Thethreechainsuperhelixhasacharacteristicrise(d)andpitch(p)asdoesthesinglechainhelix.Thecollagensuperhelixformsbecause
glycineshaveasidechainoflowstericbulk(R=H).AsthepolyprolinetypeIIhelixhasthreeresiduesperturn(n=3)andglycineisateverythirdposition,the
glycinesineachofthepolypeptidechains

 Page54

arealignedalongonesideofthehelix,forminganapolaredgeofthechain.Theglycineedgesfromthethreepolypeptidechainsassociatenoncovalentlyinaclose
arrangement,heldtogetherbyhydrophobicinteractions,toformthesuperhelixstructureofcollagen.Alargersidechainthanthatofglycinewouldimpedetheadjacent
chainsfromcomingtogetherinthesuperhelixstructure(Figure2.38).

Incollagenmoleculesthesuperhelixconformationmaypropagateforlongstretchesofthesequence,whichisespeciallytruefortypeIcollagenwhereonlythe
COOHterminalandNH2terminalsegments(knownasthetelopeptides)arenotinasuperhelicalconformation.ThetypeIcollagenmoleculehasalengthof3000
andawidthofonly15,averylongcylindricalstructure.Inothercollagentypes,thesuperhelicalregionsmaybebrokenperiodicallybyregionsofthechainthatfold
intoglobulardomains.

FormationofCovalentCrosslinksinCollagen

Anenzymepresentinextracellularspaceactsonthesecretedcollagenmolecules(seep.747)toconvertsomeofthee aminogroupsoflysinesidechainstod
aldehydes(Figure2.39).Theresultingaminoacid,containinganaldehydicRgroup,isthederivedaminoacidallysine.Thenewlyformedaldehydesidechain
spontaneouslyundergoesnucleophilicadditionreactionswithnonmodifiedlysinee aminogroupsandwiththed carbonatomsofotherallysinealdehydicgroupsto
formlinkingcovalentbonds(Figure2.39).Thesecovalentlinkagescanbebetweenchainswithinthesuperhelicalstructureorbetweenadjacentsuperhelicalcollagen
moleculesinacollagenfibril.

ElastinIsaFibrousProteinwithAllysineGeneratedCrosslinks

Elastingivestissuesandorgansthecapacitytostretchwithouttearing.Itisclassifiedasafibrousproteinbecauseofitsstructuralfunctionandrelativeinsolubility.Itis
abundantinligaments,lungs,wallsofarteries,andskin.ElastindoesnotcontainrepeatingsequencesofGlyProYorGlyXHypanddoesnotfoldintoeithera
polyprolinehelixorasuperhelix.Itappearstolackaregularsecondarystructure,butrathercontainsanunorderedcoiledstructureinwhichaminoacidresidueswithin
thefoldedstructurearehighlymobile.Thehighlymobile,kineticallyfree,thoughextensivelycrosslinkedstructuregivesthe

Figure2.39
Covalentcrosslinksformedincollagenthroughallysineintermediates.
Formationofallysinesiscatalyzedbylysylaminooxidase.

 Page55

Figure2.40
Formationofdesmosinecovalentcrosslinkinelastinfromlysineandallysines.
Polypeptidechaindrawnschematicallywithintersectionsoflinesrepresentingtheplacementof carbons.

proteinarubberlikeelasticity.Asincollagen,allysinesformcrosslinksinelastin.Anextracellularlysineaminooxidaseconvertslysinesidechainsofelastinto
allysines.TheaminooxidaseisspecificforlysinesinthesequenceLysAlaAlaLysandLysAlaAlaAlaLys.Threeallysinesandanunmodifiedlysineinthese
sequences,fromdifferentregionsinthepolypeptidechains,reacttoformtheheterocyclicstructureofdesmosineorisodesmosine.Thedesmosinescovalentlycross
linkthepolypeptidechainsinelastinfibers(Figure2.40).

a KeratinandTropomyosin

a Keratinandtropomyosinarefibrousproteinsinwhicheachchainhasana helicalconformation.a Keratinisfoundintheepidermallayerofskin,innails,andin

hair.Tropomyosinisacomponentofthethinfilamentinmuscletissue.Analysisofthea helicalsequencesinboththeseproteinsshowsthetandemrepetitionofseven
aminoacidsegments,inwhichthefirstandfourthaminoacidshavehydrophobicsidechainsandthefifthandseventhpolarsidechains.Thereiterationofhydrophobic
andpolarsidechainsinsevenaminoacidsegmentsissymbolicallyrepresentedbytheformulation(abcdefg)i,whereresiduesaanddarehydrophobicamino
acids,andresidueseandgarepolarorionizedsidechaingroups.Sinceasevenaminoacidsegmentrepresentstwocompleteturnsofana helix(n=3.6),theapolar
residuesataanddaligntoformanapolaredgealongonesideofthea helix(Figure2.41).Thisapolaredgeina keratininteractswithpolypeptideapolaredgesof
othera keratinchainstoformasuperhelicalstructurecontainingtwoorthreepolypeptidechains.Eachstrandalsocontainsapolaredge,duetoresidueseandg,that
interactswiththewatersolventontheoutsideofthesuperhelixandalsostabilizesthesuperhelicalstructure.Similarly,twotropomyosinpolypeptidestrandsina
helicalconformationwindaroundeachothertoformatropomyosinsuperhelicalstructure.

Thuscollagen,a keratin,andtropomyosinmoleculesaremultistrandstructuresinwhichpolypeptidechainswithahighlyregularsecondarystructure(polyprolinetype
IIhelixincollagen,a helixina keratinandtropomyosin)arewoundaroundeachothertoformarodshapedsupercoiledconformation.Inturn,thesupercoiled
moleculesarealignedintomultimolecularfibrilsstabilizedbycovalentcrosslinks.Theaminoacidsequencesofthechainsarerepetitive,generatingedgesonthe
cylindricalsurfacesofeachofthechainsthatstabilizeahydrophobicinteractionbetweenthechainsrequiredforgenerationofthesupercoiledconformation.

Figure2.41
Interactionofanapolaredgeoftwochains
inahelicalconformationasinakeratin
andtropomyosin.
Interactionofapolarad andda residuesof
two helicesalignedparallelinanNH2terminal
(top)toCOOHterminaldirectionispresented.
RedrawnfromMcLachlan,A.D.,andStewart,
M.J.Mol.Biol.98:293,1975.

 Page56

LipoproteinsAreComplexesofLipidswithProteins

Lipoproteinsaremulticomponentcomplexesofproteinsandlipidsthatformdistinctmolecularaggregateswithanapproximatestoichiometrybetweenproteinand
lipidcomponentswithinthecomplex.Eachtypeoflipoproteinhasacharacteristicmolecularmass,size,chemicalcomposition,density,andphysiologicalrole.The
proteinandlipidinthecomplexareheldtogetherbynoncovalentforces.

Plasmalipoproteinsareextensivelycharacterizedandchangesintheirrelativeamountsarepredictiveofatherosclerosis,amajorhumandisease(seeClin.Corr.2.5).
Theyhaveawidevarietyofrolesinbloodincludingtransportoflipidsfromtissuetotissueandparticipatinginlipidmetabolism(seeChapter9).Fourclassesof
plasmalipoproteinsexistinnormalfastinghumans(Table2.12)inthepostabsorptiveperiodafifthtype,chylomicrons,isalsopresent.Lipoproteinclassesare
identifiedbytheirdensity,asdeterminedbyultracentrifugationandbyelectrophoresis(Figure2.42).Theproteincomponentsofalipoproteinparticlearethe
apolipoproteins.Eachtypeoflipoproteinhasa

TABLE2.12HydratedDensityClassesofPlasmaLipoproteins

Lipoprotein FlotationRate,Sf MolecularWeight ParticleDiameter

Fraction Density(gmL1) (Svedbergunits) (daltons) ()
HDL 1.0631.210 HDL2,4105 70130

HDL3,2105 50100

LDL(orLDL2) 1.0191.063 012 2106 200280

IDL(orLDL1) 1.0061.019 1220 4.510 6 250

VLDL 0.951.006 20400 5106107 250750

Chylomicrons <0.95 >400 1091010 103104

Source:DatafromSoutar,A.K.,andMyant,N.B.In:R.E.Offord(Ed.),ChemistryofMacromolecules,IIB.
Baltimore,MD:UniversityParkPress,1979.

CLINICALCORRELATION2.5
Hyperlipidemias

Hyperlipidemiasaredisordersoftheratesofsynthesisorclearanceoflipoproteinsfrom
thebloodstream.Usuallytheyaredetectedbymeasuringplasmatriacylglyceroland
cholesterolandareclassifiedonthebasisofwhichclassoflipoproteinsiselevated.

TypeIhyperlipidemiaisduetoaccumulationofchylomicrons.Twogeneticformsare
known:lipoproteinlipasedeficiencyandApoCIIdeficiency.ApoCIIisrequiredby
lipoproteinlipaseforfullactivity.PatientswithtypeIhyperlipidemiahaveexceedinglyhigh
plasmatriacylglycerollevels(over1000mgdL1)andsufferfromeruptivexanthomas
(triacylglyceroldepositsintheskin)andpancreatitis.

TypeIIhyperlipidemiaischaracterizedbyelevatedLDLlevels.Mostcasesaredueto
geneticdefectsinthesynthesis,processing,orfunctionoftheLDLreceptor.
HeterozygoteshaveelevatedLDLlevelshencethetraitisdominantlyexpressed.
HomozygouspatientshaveveryhighLDLlevelsandmaysuffermyocardialinfarctions
beforeage20.

TypeIIIhyperlipidemiaisduetoabnormalitiesofApoE,whichinterferewiththeuptake
ofchylomicronandVLDLremnants.Hypothyroidismcanproduceaverysimilar
hyperlipidemia.Thesepatientshaveanincreasedriskofatherosclerosis.

TypeIVhyperlipidemiaisthecommonestabnormality.TheVLDLlevelsareincreased,
oftenduetoobesity,alcoholabuse,ordiabetes.Familialformsarealsoknownbutthe
moleculardefectisunknown.

TypeVhyperlipidemiais,liketypeI,associatedwithhighchylomicrontriacylglycerol
levels,pancreatitis,anderuptivexanthomas.

Hypercholesterolemiaalsooccursincertaintypesofliverdiseaseinwhichbiliary
excretionofcholesterolisreduced.AnabnormallipoproteincalledlipoproteinX
accumulates.Thisdisorderisnotassociatedwithincreasedcardiovasculardiseasefrom
atherosclerosis.

Havel,R.J.,andKane,J.P.Introduction:structureandmetabolismofplasma
lipoproteins.In:C.R.Scriver,A.L.Beaudet,W.S.Sly,andD.Valle(Eds.),The
MetabolicandMolecularBasisofInheritedDisease,7thed.NewYork:McGraw
Hill,1995,Chap.56andGoldstein,J.L.,Hobbs,H.H.,andBrown,M.S.Familial
hypercholesterolemia.In:C.R.Scriver,A.L.Beaudet,W.S.Sly,andD.Valle(Eds.),
TheMetabolicandMolecularBasesofInheritedDisease,7thed.,NewYork:
McGrawHill,1995,Chap.62.