Amino acids & proteins 1

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The MajorThe Major
Biomolecules ofBiomolecules of
MammalianMammalian Cells IICells II
Amino Acids & ProteinsAmino Acids & Proteins

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Part A: Amino acids (AA)Part A: Amino acids (AA)
1.1. Definitions & nomenclatureDefinitions & nomenclature
2.2. Structure & common featuresStructure & common features
3.3. ClassificationClassification

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AMINO ACIDSAMINO ACIDS
Definition:Definition:
-- organic acids containing an amine (organic acids containing an amine (––NH3) & aNH3) & a
carboxylic (carboxylic (--COOH) groupCOOH) group
-- the molecular units that make up proteinsthe molecular units that make up proteins
-- 20 naturally occurring AA are used in protein20 naturally occurring AA are used in protein
synthesissynthesis
Nomenclature:Nomenclature:
-- three letter abbreviation (e.g. Ala,three letter abbreviation (e.g. Ala, ArgArg, Asp), Asp)
-- oneone--letter symbol (e.g., A, R, D)letter symbol (e.g., A, R, D)

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Abbreviations for amino acidsAbbreviations for amino acids
Amino acid Three-letter abbreviation One-letter symbol
AlanineAlanine AlaAla AA
ArginineArginine ArgArg RR
AsparagineAsparagine AsnAsn** NN
Aspartic acidAspartic acid AspAsp DD
GlutamineGlutamine GlnGln** QQ
TryptophanTryptophan TrpTrp** WW
CysteineCysteine CysCys GG
IsoleucineIsoleucine IleIle** II
-- -- --
-- -- --
-- -- --
TyrosineTyrosine TyrTyr YY
ValineValine ValVal VV

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Structure & Common FeaturesStructure & Common Features
α-amino acid
AA as stereoisomers:AA as stereoisomers:
a centrala central αα--carboncarbon
4 different groups4 different groups
((--COOH,COOH, --NHNH33,, --H &H & --R)R)
with 4 groups onwith 4 groups on αα--C, AAC, AA
are chiral with L and Dare chiral with L and D
mirror imagesmirror images
L-ala D-ala

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molecules with mirror images are alsomolecules with mirror images are also
referred to asreferred to as stereoisomers;stereoisomers; vs.vs.
enantiomersenantiomers
most AA aremost AA are αα--amino acids & the mostamino acids & the most
commoncommon αα--AA are the LAA are the L--αα--AA.AA.
The LThe L-- and Dand D-- isomers of amino acidsisomers of amino acids

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As protein constituentsAs protein constituents::
only Lonly L--αα--AA are constituents of proteins: tooAA are constituents of proteins: too
many enantiomersmany enantiomers infinite possibleinfinite possible
combinationscombinations poorpoor stability of proteinsstability of proteins
DD--amino acids areamino acids are nevernever found in proteins,found in proteins,
although they exist in nature. Exceptions arealthough they exist in nature. Exceptions are
bacterial membrane proteins, (bacterial membrane proteins, (DD--AlaAla && DD--GluGlu))
Modified AAModified AA -- e.g. (e.g. (hydroxyhydroxy--LysLys) & () & (hydroxylhydroxyl--ProPro) in) in
collagencollagen
Occasionally there is incorporation of the rare AA,Occasionally there is incorporation of the rare AA,
selenoseleno--CysCys into few proteins using the codon UGAinto few proteins using the codon UGA

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Several common LSeveral common L--αα--AA found in cells, such asAA found in cells, such as
ornithineornithine andand citrullinecitrulline, are not used to make, are not used to make
proteins but are intermediates in the breakdownproteins but are intermediates in the breakdown
of other AAof other AA
LL--DopaDopa is an intermediate in the synthesis ofis an intermediate in the synthesis of
catecholaminescatecholamines when tyrosine iswhen tyrosine is hydroxylizedhydroxylized

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How did this particular set of AA become theHow did this particular set of AA become the
building blocks of proteins?building blocks of proteins?
1.1. They are diverse; endowing proteins with theThey are diverse; endowing proteins with the
versatility to assume many functional rolesversatility to assume many functional roles
2.2. Many of these amino acids were probablyMany of these amino acids were probably
available fromavailable from prebioticprebiotic reactionsreactions
3.3. Excessive intrinsic reactivity may have eliminatedExcessive intrinsic reactivity may have eliminated
other possible amino acids:other possible amino acids:
e.g.e.g. homohomo--SerSer && homohomo--CysCys tend to form fivetend to form five--
membered cyclic forms that limit their use inmembered cyclic forms that limit their use in
proteinsproteins

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AcidAcid--base properties:base properties:
TheThe --COOH &COOH & --NHNH22 groups in AA are capable ofgroups in AA are capable of
ionizing due to a change inionizing due to a change in pHpH..
----COOHCOOH ----COOCOO--
+ H+ H++
(3.1)(3.1)
----NHNH33
++
----NHNH22 + H+ H++
(8.0)(8.0)
At low pH (~ 1.0) bothAt low pH (~ 1.0) both ––COOH andCOOH and ––NHNH22 areare
protonatedprotonated & the molecule has net charge of +1& the molecule has net charge of +1
At pH (~ 7.4) theAt pH (~ 7.4) the ––COOH isCOOH is deprotonateddeprotonated & the& the
––NHNH22 is protonated (neutral at this pH)is protonated (neutral at this pH) zwitterionzwitterion;;
& the net charge is 0& the net charge is 0 (isoelectric point)(isoelectric point)..

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Zwitterions are substances containing equalZwitterions are substances containing equal
numbers ofnumbers of positive & negativepositive & negative charge (dipolarcharge (dipolar
ions)ions)
At higher pH both groups becomesAt higher pH both groups becomes deprotonateddeprotonated
with a net charge ofwith a net charge of --11

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pH 1 6 - 7 14
Charge + 1 0* - 1
cationcation anionanionzwitterionzwitterion
(dipole)(dipole)
pK1 pK2
Ionization State as a Function of pHIonization State as a Function of pH

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Titration curve for Ala, showing isoelectric point (Titration curve for Ala, showing isoelectric point (ppII))
Also read “ Biochem Pract. Handbook” pg 27)

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What is the major application ofWhat is the major application of pIpI??
Electrophoresis:Electrophoresis: a method of separation ofa method of separation of
proteins and other charged molecules using anproteins and other charged molecules using an
electric field (isoelectric focusing)electric field (isoelectric focusing)
Principle:Principle: a molecule with a charge will move ina molecule with a charge will move in
an electric fieldan electric field

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Classification of AA Found in ProteinsClassification of AA Found in Proteins
A: Based on the RA: Based on the R--Groups:Groups:
AA can be distinguished by RAA can be distinguished by R--groups which differgroups which differ
in:in:
-- size, shapesize, shape
-- electric chargeelectric charge
-- HH--bonding capacitybonding capacity
-- hydropathyhydropathy
-- chemical reactivitychemical reactivity

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1. With aliphatic side chains:1. With aliphatic side chains:
GlyGly is the simplest, followed byis the simplest, followed by AlaAla,, ValVal,, LeuLeu,, IIlele
The side chain ofThe side chain of IIlele becomes more extendedbecomes more extended
& more& more hydrophobichydrophobic -- cluster together rathercluster together rather
than contact waterthan contact water
ProlineProline is a cyclic but it shares many propertiesis a cyclic but it shares many properties
with the aliphatic groupwith the aliphatic group
ProlineProline influences protein architectureinfluences protein architecture
because its ring structure makes it morebecause its ring structure makes it more
conformationally restricted than the other AAconformationally restricted than the other AA

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2. Hydroxyl2. Hydroxyl--containing side chains:containing side chains:
Two AA,Two AA, SerSer && ThrThr, contain aliphatic hydroxyl, contain aliphatic hydroxyl
groupsgroups
The hydroxyl groups onThe hydroxyl groups on SerSer andand ThrThr make themmake them
much more hydrophilic (water loving) andmuch more hydrophilic (water loving) and
reactive than alanine & valinereactive than alanine & valine
SerSer can be thought of as acan be thought of as a hydroxylatedhydroxylated
version ofversion of AlaAla, whereas, whereas ThrThr resemblesresembles ValVal with awith a --
OH group in place of one of theOH group in place of one of the ValVal ––CHCH33 groupsgroups

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3.3. SulphurSulphur--containing side chains:containing side chains:
CysCys && MetMet have weak polar side chains andhave weak polar side chains and
are more hydrophilic than their aliphatic analogsare more hydrophilic than their aliphatic analogs
MetMet contains an aliphatic side chain with Scontains an aliphatic side chain with S--
atom in aatom in a thiothio--ether linkage (ether linkage (--SS--CHCH33))
CystCyst contains a sulfhydryl group (contains a sulfhydryl group (--SH). TheSH). The
sulfhydryl group is much more reactivesulfhydryl group is much more reactive
Pairs of sulfhydryl groups may come together toPairs of sulfhydryl groups may come together to
formform disulfide bondsdisulfide bonds, which are particularly, which are particularly
important in stabilizing some proteinsimportant in stabilizing some proteins

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4. Acidic side chains:4. Acidic side chains:
AspAsp && GluGlu acidacid -- the only AA that carrythe only AA that carry
negativenegative charges at pH 7 i.e., thecharges at pH 7 i.e., the --veve chargecharge inin
the side chain is retained under physiologicalthe side chain is retained under physiological
conditionsconditions
Therefore often referred to as aspartate andTherefore often referred to as aspartate and
glutamate (i.e., the conjugate bases ratherglutamate (i.e., the conjugate bases rather
than the acids)than the acids)
Uncharged derivativesUncharged derivatives AsnAsn && GlnGln with awith a
terminalterminal ––CONHCONH22 ((carboxamidecarboxamide) in place of a) in place of a
––COOH (COOH (carboxylic acidcarboxylic acid); they are both); they are both
hydrophilichydrophilic

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5. Basic side chains:5. Basic side chains:
LysLys,, ArgArg && HisHis –– relatively long side chains whichrelatively long side chains which
carrycarry positivepositive charge under physiologicalcharge under physiological
conditions (c.f. the acidic group)conditions (c.f. the acidic group)
LysLys is capped by a primary amino groupis capped by a primary amino group
whereaswhereas ArgArg has ahas a guanidiniumguanidinium groupgroup
HisHis is the least basic of the three and contains anis the least basic of the three and contains an
imidazoleimidazole group, an aromatic ring that also can begroup, an aromatic ring that also can be
positively charged. Found in many enzymes aspositively charged. Found in many enzymes as
proton donorproton donor
They are all stronglyThey are all strongly polarpolar (hydrophilic)(hydrophilic)

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6. Aromatic side chains:6. Aromatic side chains:
PhePhe, contains a, contains a phenyl ringphenyl ring attached in place ofattached in place of
one of the hydrogen atoms ofone of the hydrogen atoms of AlaAla. It is. It is
hydrophobichydrophobic
TyrTyr contains acontains a --OH group; it is the most reactive,OH group; it is the most reactive,
amino acidamino acid
TrpTrp has anhas an indoleindole ring joined to a methylenering joined to a methylene
((--CHCH22--) group) group
The aromatic AA, like most compoundsThe aromatic AA, like most compounds
carrying conjugated rings, contain delocalizedcarrying conjugated rings, contain delocalized ππ
electronselectrons that stronglythat strongly absorb light in the nearabsorb light in the near--
ultravioletultraviolet region of the spectrum at 280 nmregion of the spectrum at 280 nm

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Light absorption by aromatic amino acids at near UV region

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Note:Note: These groupings areThese groupings are
somewhatsomewhat arbitraryarbitrary and many otherand many other
sensible groupings are possible, e.g.sensible groupings are possible, e.g.
based on the electric charge of thebased on the electric charge of the
side chainsside chains

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Brain teaser!!
1.1. HOW FAR IS IT TO THE SUN?HOW FAR IS IT TO THE SUN?
2. WHY IS THE SKY BLUE?2. WHY IS THE SKY BLUE?

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B: Based on nutritional requirements:

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1. Essential amino acids:1. Essential amino acids:
Definition & FactsDefinition & Facts
These are AA thatThese are AA that MUSTMUST be provided in thebe provided in the
diet to meet an animaldiet to meet an animal’’s metabolic needs ares metabolic needs are
called essential amino acidscalled essential amino acids
Generally, EAA include those withGenerally, EAA include those with complexcomplex
structuresstructures, including some aromatic rings and, including some aromatic rings and
hydrocarbon side chainshydrocarbon side chains

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They require a large number of steps for theirThey require a large number of steps for their
synthesis and some of thesynthesis and some of the enzymesenzymes for thesefor these
steps have beensteps have been lostlost in the course of evolutionin the course of evolution
Mammals require aboutMammals require about HALFHALF of the AA inof the AA in
their diet for growth & maintenance of normal Ntheir diet for growth & maintenance of normal N--
balancebalance

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2. Non2. Non--Essential amino acids:Essential amino acids:
Definition & FactsDefinition & Facts
AA thatAA that need not be providedneed not be provided because theybecause they
cay be biosynthesized in adequate amounts arecay be biosynthesized in adequate amounts are
called nonessential amino acidscalled nonessential amino acids
These include those that areThese include those that are readily synthesizedreadily synthesized
from abundant metabolites, such as intermediatesfrom abundant metabolites, such as intermediates
in glycolysis or the citric acid cyclein glycolysis or the citric acid cycle

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Many bacteria & most plants can synthesizeMany bacteria & most plants can synthesize
all of theirall of their N metabolitesN metabolites starting from a singlestarting from a single
N source such asN source such as NHNH33 oror nitratenitrate..
Generally, preformed AA (from dietaryGenerally, preformed AA (from dietary
source) are preferred, e.g. milk dwellersource) are preferred, e.g. milk dweller
Lactobacillus spLactobacillus sp

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Amino acids classificationAmino acids classification
Light polarizationLight polarization
-- LL
-- DD
Nutritional requirementNutritional requirement
-- EAAEAA
-- NEAANEAA
Side chain/RSide chain/R--groupgroup
-- aliphaticaliphatic
-- OH groupOH group
-- acidic/basisacidic/basis
-- S contentS content
-- hydropathyhydropathy
-- etcetc ****

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Part B: ProteinsPart B: Proteins
1.1. DefinitionDefinition
2.2. General propertiesGeneral properties
3.3. The peptide bond & its formationThe peptide bond & its formation
4.4. Protein structure (1Protein structure (100 –– 4400))
5.5. Strategy to sequence a proteinStrategy to sequence a protein
6.6. Examples of different structural proteinsExamples of different structural proteins

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proteins are biopolymers (polypeptides) ofproteins are biopolymers (polypeptides) of
LL--αα--AAAA
functionally they are "functionally they are "workhorsesworkhorses" of the" of the
cell because of their many functions*cell because of their many functions*
in keeping with their functions, proteins arein keeping with their functions, proteins are
extremelyextremely complex moleculescomplex molecules

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General Properties:General Properties:
1.1. Most abundant biomolecules; accountsMost abundant biomolecules; accounts
for 50% of dry weight of most cellsfor 50% of dry weight of most cells
2.2. Built by assembling long chains of aminoBuilt by assembling long chains of amino
acids (acids (monomersmonomers), followed by intricate), followed by intricate
foldingfolding
3.3. Final shape of protein is very specific.Final shape of protein is very specific.
UnlessUnless correctly foldedcorrectly folded, is not functional, is not functional

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4.4. Several 1000Several 1000’’s different types of proteinss different types of proteins
are found in any cellare found in any cell
5.5. They areThey are mostmost structurallystructurally complexcomplex
macromolecules knownmacromolecules known
6.6. Each type of protein has its own uniqueEach type of protein has its own unique
structure and functionstructure and function

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The peptide bond (PB):The peptide bond (PB):
AA are linked together byAA are linked together by peptide bondspeptide bonds
(a.k.a. amide bond) to form polypeptide(a.k.a. amide bond) to form polypeptide
chainschains or aor a ‘‘proteinprotein’’ moleculemolecule
Proteins may consist ofProteins may consist of more than onemore than one
polypeptide chainpolypeptide chain

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PB form in the process of translation (PB form in the process of translation (proteinprotein
synthesissynthesis) when the) when the αα--NHNH33 of one AA residueof one AA residue
forms aforms a covalentcovalent bond with thebond with the αα--COOH ofCOOH of
anotheranother
----αα--COOHCOOH ++ αα--NHNH33----
HH22O is eliminated; theO is eliminated; the --C=O & theC=O & the --NN--HH
bonds are nearlybonds are nearly parallelparallel & that the C, O, N,& that the C, O, N,
and H atoms are usuallyand H atoms are usually coplanarcoplanar

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Monomer 1Monomer 1 Monomer 2Monomer 2

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Residue 2Residue 2
Residue 1Residue 1

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littlelittle twistingtwisting possible aroundpossible around
the Cthe C--N bond because the PBN bond because the PB
has doublehas double--bond character i.e.bond character i.e.
((metastablemetastable))
the peptide bond can bethe peptide bond can be
considered aconsidered a resonance hybridresonance hybrid
of the two bondsof the two bonds
atoms about the peptideatoms about the peptide
bond can exist in either thebond can exist in either the
trans*trans* oror ciscis configurationsconfigurations

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Formation the polypeptide bond:Formation the polypeptide bond:
In aqueous environment PB formation requiresIn aqueous environment PB formation requires
energyenergy ((∆∆GG ~~ +10 kJ/mol, RT)+10 kJ/mol, RT);; therefore not favoredtherefore not favored
(c.f. hydrolysis)(c.f. hydrolysis)
AA has to be activated and then attached to aAA has to be activated and then attached to a
transfer RNA (tRNA)transfer RNA (tRNA)
ATP is hydrolyzed to AMP &ATP is hydrolyzed to AMP & PPiPPi and the bond isand the bond is
formed betweenformed between ––COOH & 3'COOH & 3'--OH of the adenosineOH of the adenosine
of tRNA moleculeof tRNA molecule aminoacylaminoacyl--tRNAtRNA

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Activated AA
Activation of amino acidActivation of amino acid **

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Protein Structure & OrganizationProtein Structure & Organization
Protein molecules haveProtein molecules have fourfour levels oflevels of
structural organization:structural organization:
-- Primary (or amino acid sequence)Primary (or amino acid sequence)
-- Secondary (or local regular folding)Secondary (or local regular folding)
-- Tertiary (or overall folding)Tertiary (or overall folding)
-- Quaternary (multiQuaternary (multi--chain association)chain association)

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The primary structure (1The primary structure (1°°))
•• Refers to the unique & definedRefers to the unique & defined sequence of AAsequence of AA inin
a protein moleculea protein molecule
•• Depending on the characteristics of AA, the 1Depending on the characteristics of AA, the 1°°
determines the properties and shape of the proteindetermines the properties and shape of the protein
functionfunction

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•• Proteins evolve over time thruProteins evolve over time thru changeschanges in their AAin their AA
sequences:sequences:
-- conservative e.g.,conservative e.g., AspAsp vs.vs. GluGlu
-- nonnon--conservative e.g.,conservative e.g., AspAsp vs.vs. AlaAla
•• Determined by the nucleotide base sequence ofDetermined by the nucleotide base sequence of
DNA (DNA (genetic codegenetic code); not just a random sequence); not just a random sequence
(chances(chances -- 1 in 201 in 20500500
))

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The primary structure of bovine insulin

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Determination of primary structureDetermination of primary structure
(protein sequencing)(protein sequencing)
-- determine the number of polypeptide ordetermine the number of polypeptide or
subunitssubunits
-- cleave the disulphide bondscleave the disulphide bonds
-- separate subunits and purifyseparate subunits and purify
-- determine component AA usingdetermine component AA using EdmanEdman
reactionreaction & ion exchange chromatography& ion exchange chromatography

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Edman degradation reactionEdman degradation reaction
•• A rA reaction in whicheaction in which AAAA residues are removed fromresidues are removed from
the polypeptide chainthe polypeptide chain one by oneone by one at a time, startingat a time, starting
from Nfrom N--terminus (automated systems)terminus (automated systems)
The reaction:The reaction:
•• PhenylPhenyl--isoiso--thiothio--cyanatecyanate (PITC)(PITC) reacts with the AAreacts with the AA
residue at the Nresidue at the N--terminus under basic conditions toterminus under basic conditions to
form aform a PITCPITC--proteinprotein derivativederivative

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•• TrifluoroaceticTrifluoroacetic acid (acid (TFATFA) then) then cleavescleaves off theoff the
first AA as itsfirst AA as its anilinoanilino--thiolinonethiolinone derivative (derivative (ATZATZ--
amino acidamino acid) and leaves the new N) and leaves the new N--terminus forterminus for
the next degradation cyclethe next degradation cycle
•• Using NUsing N--butyl chloride and with 25% TFA/water,butyl chloride and with 25% TFA/water,
thethe ATZATZ--aminoamino acid is thenacid is then convertedconverted to ato a
phenylphenyl--thiothio--hydantoinhydantoin derivative (derivative (PTHPTH--aminoamino
acidacid))

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•• The PTHThe PTH--amino acid is transferred to a reverseamino acid is transferred to a reverse--
phasephase HPLC columnHPLC column for detection at 270nm; a stdfor detection at 270nm; a std
mixture of AA is usedmixture of AA is used
•• StdStd retention timesretention times of the AAof the AA comparedcompared with eachwith each
Edman degradation cycle chromatogramEdman degradation cycle chromatogram
•• The HPLC chromatograms are analyzed using aThe HPLC chromatograms are analyzed using a
computercomputer data analysis systemdata analysis system

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Residue 1: Leucine
Std cycle
Residue 2: Isoleucine
PerkinPerkin Elmer AppliedElmer Applied
BiosystemsBiosystems protein/peptideprotein/peptide
sequencersequencer

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The Secondary Structure (2The Secondary Structure (2°°))
•• TheThe spatialspatial arrangement of AA residuesarrangement of AA residues
that are near one another in the linearthat are near one another in the linear
sequence i.e.sequence i.e. local regular foldinglocal regular folding
•• In theIn the myoglobinmyoglobin
molecule the chainmolecule the chain
appears to be locallyappears to be locally
coiled into regions ofcoiled into regions of
helicalhelical structuresstructures
•• PaulingPauling: showed most: showed most
22°° structures are thestructures are the αα--
helixhelix and theand the ββ--strandsstrands

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Helix repeat
n = m/t
m = number of residues per repeat (res) 18
t = number of turns per repeat (turn) 5
n =number of residues per turn (res/turn) 18/5 = 3.6

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Helix pitch
P = nh
P = pitch (nm/turn)
h = rise (nm/res)
n = number of residue per turn (res/turn)

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1.1. αα helixhelix::
PP chain twists into aPP chain twists into a
tightly packed rod; thetightly packed rod; the
HH-- bondsbonds (NH(NH——COOCOO--))
areare withinwithin a single PPa single PP
chain parallel to thechain parallel to the
helix axishelix axis
2.2. Beta sheet:Beta sheet:
PP chain is nearly fullyPP chain is nearly fully
extended; the Hextended; the H--
bonds arebonds are betweenbetween thethe
adjacent chains (NHadjacent chains (NH——
COOCOO--) & nearly) & nearly
perpendicularperpendicular to theto the
chainschains
α-helix β-sheet

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i.i. The keratinsThe keratins --
αα--keratinskeratins they are coiledthey are coiled
αα--helical and found in skin,helical and found in skin,
hair & nailshair & nails
ββ--keratinskeratins found mainly infound mainly in
birds and reptiles; they arebirds and reptiles; they are
ββ--sheetsheet
Fibrous proteinsFibrous proteins are specificare specific
examples of proteins with 2examples of proteins with 2°°
structure and they usually playstructure and they usually play
structural roles in the cell
Proteins with 2Proteins with 2°° StructureStructure
structural roles in the cell

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Proposed structureProposed structure
for keratin typefor keratin type
filamentsfilaments

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ii.ii. FibroinFibroin --
-- aa ββ--sheetsheet protein spun by silkworm and spiders;protein spun by silkworm and spiders;
half of its AA residues arehalf of its AA residues are GlyGly with a fewwith a few AlaAla && SerSer
-- this sequence allows the sheets to fit and packthis sequence allows the sheets to fit and pack
on top of one anotheron top of one another
-- resulting in a fiber that is strong and relativelyresulting in a fiber that is strong and relatively
inextensibleinextensible

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[[GlyGly--AlaAla--GlyGly--AlaAla--GlyGly--SerSer--GlyGly--AlaAla--AlaAla--GlyGly--(Ser(Ser--GlyGly--AlaAla--GlyGly--AlaAla--GlyGly))88]]

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iii.iii. CollagenCollagen ––
-- most abundant inmost abundant in
vertebrates; ~1/3; it is forvertebrates; ~1/3; it is for
strength & toughness (strength & toughness (bone &bone &
tendons, skintendons, skin))
-- basic unitbasic unit tropotropo--collagencollagen
molecule, a triplemolecule, a triple helixhelix ofof
three polypeptide chainsthree polypeptide chains
(rich in(rich in GlyGly andand ProPro, each ~, each ~
1000 AA1000 AA
-- ProPro in the collagen fiber arein the collagen fiber are
mostly modified tomostly modified to hydroxyhydroxy--
proline**proline** to stabilize theto stabilize the
structurestructure

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CollagenCollagen & vitamin C& vitamin C
-- the enzyme catalyzingthe enzyme catalyzing hydroxylationhydroxylation
of Proline requiresof Proline requires ascorbicascorbic acidacid
-- severe vitamin C deficiency leads tosevere vitamin C deficiency leads to
scurvyscurvy
-- the condition recoversthe condition recovers quicklyquickly afterafter
administration of the vitaminadministration of the vitamin

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iv.iv. ElastinElastin ––
-- has ahas a random coil structurerandom coil structure with little of 2with little of 2°°
-- required for highly elastic tissues e.g. arterialrequired for highly elastic tissues e.g. arterial
bloodblood vesselsvessels, &, & ligamentsligaments
-- the PP chain is rich inthe PP chain is rich in GlyGly,, AlaAla && ValVal
-- to prevent the fibers from indefinite extensionto prevent the fibers from indefinite extension
the polypeptide chain has a fewthe polypeptide chain has a few LysLys residues forresidues for
crosscross--linkagelinkage

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The Tertiary Structure (3The Tertiary Structure (3°°))
•• The 2The 2°° themselves are in turn foldedthemselves are in turn folded
into a specificinto a specific compactcompact structure forstructure for
the entire polypeptide chainthe entire polypeptide chain
•• Tertiary structures areTertiary structures are
found infound in globular proteinsglobular proteins;;
they are responsible forthey are responsible for
most of the work in themost of the work in the
cellcell –– transport,transport,
metabolic, biosynthetic,metabolic, biosynthetic,
etcetc

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Examples of 3Examples of 3oo
proteins:proteins:
i.i. MyoglobinMyoglobin --
-- has 70%has 70% αα--helix with a prosthetic group (helix with a prosthetic group (hemeheme))
ii.ii. Bovine pancreatic trypsin inhibitor (BPTI)Bovine pancreatic trypsin inhibitor (BPTI) --
-- smallest globular protein (smallest globular protein (58 AA58 AA).).
-- mostlymostly ββ--sheetsheet structure connected by bends instructure connected by bends in
the chainthe chain
-- sole function is to bind to and inhibit proteolyticsole function is to bind to and inhibit proteolytic
enzymeenzyme trypsintrypsin to preventto prevent autocatalysisautocatalysis ofof
pancreaspancreas
-- threethree disulphidedisulphide bonds are also foundbonds are also found

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iii.iii. Larger globular proteinsLarger globular proteins --
-- these are composed of multiplethese are composed of multiple domainsdomains i.e.,i.e.,
compact folded 3compact folded 3°° structuresstructures
-- domains are connected by specific polypeptidedomains are connected by specific polypeptide
strandsstrands that run thru the moleculethat run thru the molecule
-- all GP have definedall GP have defined insideinside andand outsideoutside
(hydrophobic & hydrophilic residues respectively)(hydrophobic & hydrophilic residues respectively)

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The Quaternary Structure (4The Quaternary Structure (4°°))
•• The association of PP chains to form specificThe association of PP chains to form specific multimulti--
subunitsubunit structures; as most proteins in the cell existstructures; as most proteins in the cell exist
Levels of 4Levels of 4°° organizationorganization
1.1. HomotypicHomotypic --
–– identical or nearly identical PP chains e.g.identical or nearly identical PP chains e.g.
hemoglobin (hemoglobin (HbHb),), a tetramer of myoglobina tetramer of myoglobin--likelike
chainschains

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2.2. HeterotypicHeterotypic ––
-- interaction between subunits of very differentinteraction between subunits of very different
structures e.g.structures e.g. BPTIBPTI interaction withinteraction with trypsin:trypsin:
-- the two protein surfaces fit one another closelythe two protein surfaces fit one another closely
to form a specific complexto form a specific complex
-- some are very complex e.g.some are very complex e.g. PyruvatePyruvate
dehydrogenasedehydrogenase with 72 subunitswith 72 subunits

88
Complex Protein StructuresComplex Protein Structures
•• Proteins covalentlyProteins covalently conjugated with otherconjugated with other
moleculesmolecules; this occurs as post; this occurs as post--translationaltranslational
modificationmodification
Examples of complex proteins:Examples of complex proteins:
i.i. GlycoproteinsGlycoproteins –– associated with CHassociated with CH22OO; found on; found on
the surface of RBC are extremely important asthe surface of RBC are extremely important as
they determine blood group specificitiesthey determine blood group specificities
ii.ii. LipoproteinsLipoproteins -- proteins associated withproteins associated with lipidslipids viavia
nonnon--covalent interactions (transport & storagecovalent interactions (transport & storage
of lipid and cholesterol)of lipid and cholesterol)

89
iii.iii. PhosphoproteinsPhosphoproteins –– associated with phosphateassociated with phosphate
groupgroup; e.g., milk protein, casein; e.g., milk protein, casein
iv.iv. MetalloproteinsMetalloproteins -- proteins associated withproteins associated with metalmetal
e.g.,e.g., ferritinferritin (Fe),(Fe), calmodulincalmodulin (Ca),(Ca),
ceruloplasminceruloplasmin (Cu), etc(Cu), etc

93
Forces Controlling Protein StructureForces Controlling Protein Structure
1.1. Hydrogen BondingHydrogen Bonding --
•• Interaction between a covalently bondedInteraction between a covalently bonded H atomH atom
on aon a donordonor groupgroup (e.g.(e.g. --OH) & a pair of nonOH) & a pair of non--
bondedbonded electrons on an acceptorelectrons on an acceptor group e.g. O / Ngroup e.g. O / N
•• In proteins, HIn proteins, H--bonding, occurs not onlybonding, occurs not only withinwithin andand
betweenbetween peptide chains but also with thepeptide chains but also with the
surroundingsurrounding aqueous mediumaqueous medium
•• strongest ones being those in which the donor, Hstrongest ones being those in which the donor, H
and acceptor atoms areand acceptor atoms are collinearcollinear. E.g., in the. E.g., in the αα--
helix in proteins & the DNA double helixhelix in proteins & the DNA double helix

95
2.2. HydrophathicHydrophathic ForcesForces --
•• HH--bondingbonding between hydrophilic Rbetween hydrophilic R--groups andgroups and
the aqueous environmentthe aqueous environment
•• RepulsionRepulsion from the aqueous environment by thefrom the aqueous environment by the
hydrophobic Rhydrophobic R--groups (& vice versa)groups (& vice versa)
•• All these forcesAll these forces stabilizesstabilizes the protein moleculethe protein molecule

96
3. Electrostatic Forces3. Electrostatic Forces --
•• chargecharge--chargecharge –– between oppositely charged Rbetween oppositely charged R--
groupsgroups
•• charge dipolecharge dipole -- the interaction of ionized Rthe interaction of ionized R--groupsgroups
of AA with the dipole of the Hof AA with the dipole of the H22OO
•• dipoledipole--dipoledipole -- the slight dipole moment that existsthe slight dipole moment that exists
in the polar Rin the polar R--groups of AA also influences theirgroups of AA also influences their
interaction with Hinteraction with H22OO

98
4. van4. van derder WaaalsWaaals ForcesForces --
Very weak attractive & repulsive forces:Very weak attractive & repulsive forces:
•• AttractiveAttractive –– among induced dipoles that ariseamong induced dipoles that arise
from fluctuations in the charge densities thatfrom fluctuations in the charge densities that
occur between adjacent uncharged nonoccur between adjacent uncharged non--
bonded atomsbonded atoms
•• RepulsiveRepulsive -- occur when uncharged nonoccur when uncharged non--
bonded atoms come very close together but dobonded atoms come very close together but do
not induce dipoles (i.e., electron repulsion)not induce dipoles (i.e., electron repulsion)

101
Denaturation & Renaturation of ProteinsDenaturation & Renaturation of Proteins
DenaturationDenaturation
•• Disruption and possible destruction of both theDisruption and possible destruction of both the
secondarysecondary andand tertiarytertiary structures leading to unfoldingstructures leading to unfolding
and loss of function (and loss of function ( coagulationcoagulation && precipitationprecipitation))
•• The 1The 1°° (sequence of AA)(sequence of AA) remains the sameremains the same after aafter a
denaturation processdenaturation process
•• When the 2When the 2°° and 3and 3°° structures are lost thestructures are lost the
polypeptide becomespolypeptide becomes a random coila random coil

103
RenaturationRenaturation
•• = reversible denaturation process= reversible denaturation process
•• Sometimes an unfolded protein can be restored toSometimes an unfolded protein can be restored to
correct folding andcorrect folding and regainregain biological activitybiological activity
•• Irreversible as when egg white protein or albuminIrreversible as when egg white protein or albumin
is denatured by boilingis denatured by boiling
•• Renaturation was first demonstrated byRenaturation was first demonstrated by ChristianChristian
AnfinsenAnfinsen ““AnfinsenAnfinsen experimentexperiment””

104
Ribonuclease
(active)
Denatured
Ribonuclease
(inactive)
B-mercaptol-ethanol
and 8M urea
Ribonuclease
(active)
B-mercaptol-ethanol
and 8M urea
AnfinsenAnfinsen experimentexperiment

105
Denaturation agentsDenaturation agents
•• HeatHeat -- heat increases the kinetic energy andheat increases the kinetic energy and
causes the molecules to vibrate so rapidly andcauses the molecules to vibrate so rapidly and
violently that the bonds are disruptedviolently that the bonds are disrupted
•• AlcoholAlcohol –– it disrupts Hit disrupts H--bonding. E.g. a 70%bonding. E.g. a 70%
alcohol solution for disinfectionalcohol solution for disinfection
•• AcidAcid andand basesbases –– they disrupt salt bridges (ionicthey disrupt salt bridges (ionic
bonds) which result from the neutralization of anbonds) which result from the neutralization of an
acid and amine on side chainsacid and amine on side chains

106
•• Heavy metal saltsHeavy metal salts –– usually contain Hgusually contain Hg+2+2
, Pb, Pb+2+2
,,
AgAg+1+1
TlTl+1+1
, Cd, Cd+2+2
etc with high atomic weightsetc with high atomic weights
disrupt salt bridgesdisrupt salt bridges
•• Reducing agentsReducing agents –– they act to denature proteinsthey act to denature proteins
by disrupting disulfide bonds. Reducing agentsby disrupting disulfide bonds. Reducing agents
add hydrogen atoms to make theadd hydrogen atoms to make the thiolthiol group,group, --SHSH

Amino acids & proteins 1

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