An introduction to proteins and peptides · An introduction to proteins and peptides The fundamental component of a protein is the polypeptide chain composed of ... this gives a blue

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An introduction to proteins andpeptides

The fundamental component of a protein is the polypeptide chain composed ofamino acid residues; twenty different residues are involved in protein synthesis.These residues might be modified after the synthesis of the polypeptide chain.The other components of proteins are called prosthetic groups.The structure ofthe amino acids and their characteristic property as amphoteric molecules isdescribed, followed by a description of asymmetry and chirality.The way inwhich amino acid residues interact within proteins is explained.The ionicproperties of proteins are important in such interactions and in theirelectrophoretic separation. Proteins can also be separated on the basis of their size.After mentioning how the order of the amino acid residues in polypeptides canbe determined, the hierarchies of protein structure are briefly described.Thetertiary structure of proteins can be destroyed by denaturation. Finally, it is shownthat even small peptides can possess biological activity, for example as hormonesand transmitters.

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THE ROLE OF AMINO ACIDS INTHE CELL

Amino acids are a fine example of theversatile roles performed by the cellconstituents.Amino acids contain, amongother functional groups, two that arecommon to all amino acids: an amino (orimino) group and a carboxyl group.Theability of an amino acid to condense withother amino acids to form a peptide isdependent on the chemical properties ofthese two functional groups. Certainly, amost important role for amino acids is toserve as the monomeric subunits ofproteins, but they have other importantroles. For example, the tripeptideglutathione has an important function andother small peptides serve as hormonesand, in some organisms, as antibiotics;glutamic acid acts as a neural transmitter.Amino acids are the precursors of a widevariety of biomolecules (e.g. nitric oxidefrom arginine, histamine from histidine).Some amino acids are metabolized andutilized for the production of glucose(gluconeogenesis).As there is no store ofamino acids, apart from those involved inprotein structure, proteins have to bebroken down to free amino acids when thelatter are required for gluconeogenesis.

STRUCTURE OF AMINO ACIDS

All the common amino acids, except forproline, have the same general structure inthat the α-carbon atom bears a –COOHgroup, an –NH2 group and an ‘R’-group,which is responsible for the differentproperties of the various amino acids.A general formula for amino acids isshown in Fig. 2.1.The structures of the 20 common amino acids are shown in Fig. 2.2, grouped according to the natureof their R-groups.The internationally-approved three-letter and single-letterabbreviations for each amino acid are alsoindicated.

The α carbon is optically active in α-amino acids other than glycine.The twopossible isomers are termed D and L.Allnaturally occurring amino acids found inproteins are of the L-configuration (see p. 9).

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R

+H3NCHCOO–

Fig. 2.1 General formula of an amino acid.

1. Non-polar or hydrophobic R-groups

2. Negatively charged R-groups at pH 6–7

3. Uncharged or hydrophilic R-groups

4. Positively charged R-groups at pH 6–7

Fig. 2.2 Structures of the 20 common amino acids grouped according to the nature of their ‘R’-group. Note the three- and one-letter notations.

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A cystine residue is formed from twocysteines linked through a disulfide bridge(–S–S–) formed from their sulfhydryl(–SH) groups.

The charges on the amino acidsindicated in Fig. 2.2 are those that occur at pH 6–7.Acids are defined as protondonors and bases as proton acceptors.It follows that, at pH 6–7, an amino acid ingroup 2 is present as a free base (an anion)and one in group 4 as a free acid (a cation).The terms ‘acidic’ and ‘basic’, asapplied to amino acids, should therefore beused with caution because they refer to theprotonated forms of group 2 or theunprotonated forms of group 4.Acompound such as an amino acid thatcarries both basic and acidic groups isreferred to as amphoteric.

ASYMMETRY IN BIOCHEMISTRY

ASYMMETRY AS APPLIED TOAMINO ACIDS AS AN EXAMPLE

Chirality is derived from the Greek wordcheir for ‘hand’ – the left and right handsare mirror images of each other. Suchasymmetry in molecular structure is ofgreat importance in biochemistry.A chiralmolecule possesses at least one asymmetriccentre, such as a carbon atom, to which arejoined four groups that are different fromeach other.

The amino acid alanine can exist in twoforms, denoted D-alanine and L-alanine, asshown in Fig. 2.3.The amino acidscontained in mammalian proteins are ofthe L-form. (Sugars are also chiralmolecules; D-sugars predominate inmammalian carbohydrates; see p. 110.) InFig. 2.3, red denotes the oxygen atoms ofthe carboxyl group, the nitrogen atom ofthe amino acid group is grey, the carbonatoms are black and the hydrogen atomsare white.

NON-CHIRAL ASYMMETRY

Even if a molecule is not chiral, it cancontain identical groups that are stericallydistinguishable.A simplified representationof a hypothetical molecule is shown in Fig. 2.4. If A and B are held in space on asurface, then the identical groups X1 andX2 can be distinguished.The classicbiochemical example is citric acid.Although this molecule has a plane ofsymmetry, the central carboxyl group andthe hydroxyl group can be held in such away that the two –CH2COOH groups canbe distinguished and the molecule is able

to interact with an enzyme that hasspecific binding sites for the differentgroups in the molecule (see Fig. 9.15,p. 136). Such a molecule is termed‘prochiral’ in that it can be made chiral bychanging the structure of the group ononly one of the central carbon bonds.Note that, if a molecule has a plane ofsymmetry such that chiral centres on eitherside of the plane of symmetry exactlycompensate, the molecule is termed a mesocompound (e.g. meso-tartaric acid, shownin Fig. 2.4).

R AND S CONVENTION

A chiral centre can be denoted R or S.The method for ascribing the R or Sdesignation to a centre is as follows:

• List the functional groups in order oftheir priority assigned by convention.The order for some biochemicallyimportant groups is –SH (highest),–OH, –NH2, –COOH, –CHO, –CH3,–H (lowest).Then orientate themolecule so that the group of lowestpriority points away from the observer.

• If the order of priority (high to low) ofthe remaining groups is clockwise, thecentre is R. If the order or priority isanticlockwise, the centre is S.Thus theα-carbon of L-alanine has the Sconfiguration.

IONIC PROPERTIES OF AMINOACIDS

ELECTROPHORETIC SEPARATION

As already explained, amino acids haveamphoteric properties that allow theirseparation by electrophoresis at pH 6.0,in which the amino acids move along amedium (paper) under the force of anapplied electric field. Such a separation isillustrated in Fig. 2.5. Electrophoresis iscommonly carried out on paper but gelscan also be used.The amphoteric nature ofα-amino acids means that, in the absence

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Fig. 2.3 D- and L-alanine are examplesof asymmetry instructure.

Fig. 2.4 Example of a meso compound (left)and the simple representation of ahypothetical molecule (right).

Fig. 2.5 Demonstration ofthe ionic properties ofamino acids byelectrophoresis.

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of other acids or bases, the carboxyl andamino groups are both fully ionized, givingrise to the term zwitterion (GermanZwitter = hybrid or hermaphrodite).This isthe form that predominates in neutralsolution and in crystals, rather than theunionized form.

THE BUFFERING CAPACITY OFAMINO ACIDS

As explained previously, an acid is definedas a proton donor.Acids vary in theirtendency to dissociate; stronger acids do somore readily than weaker ones.Thestrength of an acid is expressed by the termpKa, which is the pH at which an acid is50% dissociated.The titration curve ofalanine (Fig. 2.6) shows that the –COOHgroup becomes more dissociated as the pHincreases.At pK1, the change in pH of thesolution with increasing additions ofNaOH is lowest; in other words, thebuffering capacity is greatest.We have defined a base as a protonacceptor; so, in this case, the proportion ofionized NH3 decreases as the pH increasesand the maximum buffering is at pK2.

The buffering capacity of histidine

If the R-group of an amino acid is capableof being ionized, then the amino acid willhave a third pK. Histidine is veryimportant in this respect because theimidazole group is only weakly basic,having a pKa of 6.00. It therefore exists as amixture of the protonated and dissociatedforms in solution at the physiological pHof 7.2–7.4. Histidine therefore contributesto the buffering capacity of proteins.Thetitration curve of histidine is shown in Fig. 2.7.

SEPARATION OF AMINO ACIDS BY ION-EXCHANGECHROMATOGRAPHY

It is often important to determine theproportion of the different amino acids,either in body fluids such as serum orspinal fluid, or in a protein hydrolysate.For this purpose, a resin bearing eitherpositively charged groups (anion-exchangeresin) or negatively charged groups (cation-exchange resin) can be used.Amino acidspassed down a column of such a resin bindcompetitively to the charged groups on theresin.

Figure 2.8 shows the separation of theamino acids present in a peptidehydrolysate on a column of sulfonated

polystyrene (cation-exchange resin).Passage through the column of buffers ofincreasing pH causes aspartic acid (acidic)to emerge as the first amino acid andarginine (basic) as the last. It is common todetect the amino acids using ninhydrin;

this gives a blue colour after reaction withall α-amino acids (yellow for the aminoacid proline), the intensity of colour beingrelated to the amount of the particularamino acid.The whole process can beautomated.

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Fig. 2.6 Amino acids possess bufferingcapacity, as demonstrated by thetitration of alanine.

Fig. 2.7 The bufferingproperties of histidineare of particularphysiologicalimportance.

Fig. 2.8 Amino acids can beseparated by ion-exchangechromatography and theamount of each determined.

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Similar methods can be used for theseparation of proteins that carry variousnet charges.

PEPTIDE STRUCTURE AND THE PEPTIDE BOND

THE PEPTIDE BOND

The peptide bond is formed by theinteraction of two amino acids, with theelimination of water between theneighbouring –NH2 and –COOH groups.This is shown in Fig. 2.9.The peptidebond is a rigid structure; this has importantimplications for the structure of proteins(see p. 56).

Proline can also participate in a peptidebond (Fig. 2.10) but, in contrast to the α-amino acids, there is then no H availablefor H bonding which, as we will see, isimportant in the secondary structure ofproteins.

NOTATION USED FOR PEPTIDES

The structure of a typical peptide,enkephalin, is shown in Fig. 2.11. Inwriting the primary structure, one startswith the amino-terminus (also called theN-terminus) and ends with the carboxy-terminus (referred to as the C-terminus).In Fig. 2.11, enkephalin is given in thethree-letter code for amino acids.Abbreviated according to the single-lettercode it would be written YGGFM.A peptide composed of more than a fewamino acid residues is termed apolypeptide.To the extent that suchpolypeptides are the backbone structures of

proteins, there is no formal definition of atransition from polypeptide to protein, butinsulin, which has 50 amino acid residues,is commonly regarded as being typical ofthe smallest protein.

IDENTIFICATION OF PEPTIDE BONDS

The presence of a peptide bond is usuallydetermined by the biuret reaction. Biurethas the formula NH2CONHCONH2 andis a simple substance possessing a peptidebond.When biuret is treated with CuSO4

in alkaline solution, a purple colour isproduced.This is known as the biuretreaction and, as expected, proteins give astrong reaction.

IONIC PROPERTIES OF PEPTIDES

THE NATURE OF THE CHARGED R-GROUPS

The ionizable, dissociable α-amino and α-carboxyl groups of the amino acids areblocked by peptide formation, except forthe terminal residues.The ionized state of aprotein therefore depends almost entirelyon the R-groups; this, in effect, meansthose on aspartic and glutamic acids, lysine,arginine and histidine.This is illustrated inFig. 2.12, which shows the structure of ahypothetical peptide containing all thesegroups.The numbers indicate the pK rangeof each dissociating group.As indicatedabove, histidine is very important becauseits charge can vary over the physiologicalpH range.

THE ISOELECTRIC POINTS OFPROTEINS

The isoionic point is the pH that resultswhen the protein, freed of all other ions, isdissolved in water.The isoelectric point is

the pH at which there is zero migration inan electric field (see below) to eitherelectrode.The isoelectric points of a rangeof proteins are shown in Table 2.1. On thebasis of these values, proteins are describedas basic, neutral or acidic, depending onwhether their overall charge atphysiological pH is positive, approximatelyzero or negative.

ELECTROPHORESIS OF PROTEINS

Just as amino acids can be separated byelectrophoresis so can proteins. Figure 2.13shows the result of the electrophoresis ofhuman serum proteins on a cellulose strip(paper can also be used) in a buffer at pH 8.6.The separated protein bands arevisualized after staining with dye, and adensitometric scan provides an indicationof the relative amount of protein in each

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Fig. 2.9 Peptide bonds are formed by theinteraction of amino acids.

Fig. 2.10 The participation of proline in apeptide bond.

Fig. 2.11 The structure of a typical peptide.

Fig. 2.12 Polypeptidespossess ionicproperties, mainly dueto the R-groups on theamino acid residues.

Table 2.1 The isoelectric points of somecommon proteins

Protein Isoelectric point

Blood proteins

α1-Globulin 2.0Haptoglobin 4.1Serum albumin 4.7γ1-Globulin 5.8Fibrinogen 5.8Hemoglobin 7.2γ2-Globulin 7.4

Miscellaneous proteins

Pepsin 1.0Ovalbumin 4.6Insulin 5.4Histones 7.5–11.0Ribonuclease 9.6Cytochrome c 9.8Lysozyme 11.1

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band. ‘S’ indicates the point of applicationof the serum before applying the currentwith the charges shown.Although themobility of the proteins depends mainly ontheir relative charge, the size of theproteins also plays a part and this certainlycontributes to the position of the large γ-globulin band.Although the serumproteins give the appearance of beingseparated into discrete bands, it should beremembered that, with the exception ofserum albumin, each band contains manydifferent proteins.

POLYACRYLAMIDE GELELECTROPHORESIS (PAGE) OFPROTEINS

Polyacrylamide gel can be used instead ofcellulose acetate or paper for the separationof native proteins. Such a gel is commonlyused in the presence of sodium dodecylsulfate (SDS). In this case, oligomericproteins (those composed of severaldiscrete polypeptides) are separated in theform of their subunits.

Figure 2.14 shows the resolution ofproteins by SDS-PAGE.The proteins aresuspended in a 1% solution of SDS.This

detergent disrupts most protein–proteinand protein–lipid interactions.Very often,2-mercaptoethanol is also added, to disruptdisulfide bonds.The electrophoreticmobility of most proteins, but notglycoproteins, depends on their size, as thenegative charge contributed by SDSmolecules bound to the protein is muchlarger than the net charge of the proteinitself.A pattern of bands appears when thegel is stained with Coomassie Blue.

Agarose can be used in place ofpolyacrylamide gel for larger proteins or toobtain a different type of separation in theabsence of SDS.

Two-dimensional PAGE can also becarried out, using different conditions ineach direction: for example, animmobilized pH gradient (pH 4–7) in onedirection and an 11–14% polyacrylamidegradient in the other.

NON-COVALENT BONDS INPROTEINS

The distribution of charged amino acidgroups in a polypeptide chain has alreadybeen described.The charged groups areimportant in terms of the folding of the

chains because negatively charged groupswill repel each other, as will positivelycharged groups, whereas closely positionednegative and positive charges will attracteach other.There are, however, severalother important interactions between theR-groups in proteins.These are illustratedin Fig. 2.15.

The ionic interactions already referredto are also known as salt bridges and areillustrated by an interaction betweenglutamate and arginine.The S–S bondsformed by the oxidation of two sulfhydrylgroups are covalent and are particularlylikely to be present in proteins when thephysiological environment is unfriendly;they enhance the rigidity of the protein.An example is the proteins in the digestivesecretions of the pancreas.

The other interactions are described as non-covalent and can be either apolar(i.e. hydrophobic) or polar (i.e. ionic andhydrogen bonding). Hydrophobicinteractions result from: (i) van der Waalsinteractions, which arise from an attractionbetween atoms due to fluctuating electricdipoles originating from the electroniccloud and positive nucleus; (ii) thehydrophobic effect, which is the tendencyof non-polar groups to associate with oneanother rather than to be in contact withwater. Hydrogen bonds arise because, whena hydrogen atom is linked to an oxygenatom, there is a shift of electrons leading toa partial negative charge on the otheratom.This produces an electric dipole thatcan interact with dipoles that existelsewhere.The most common hydrogenbond is between –N–H and –C=O, as inthe α helix and β-pleated sheet (to bedescribed in Chapter 4), but other bondsare possible, as shown in Fig. 2.15.

PURIFICATION OF PROTEINS ANDDETERMINATION OF RELATIVEMOLECULAR MASS

PURITY AND HOMOGENEITY

The purification of small molecules hastraditionally ended with crystallization andthe determination of various physicalparameters, such as the melting point, butthese procedures are much less applicableto macromolecules such as proteins. Even ifproteins are crystallized, they might becontaminated by other proteins, by virusesor by other infective agents such as prions(see p. 60).The objective in thepurification of proteins, therefore, is toproduce a product that is homogeneous byall known criteria, which usually includes

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Am

ount

of p

rote

in

Fig. 2.13 Separation of serum proteins byelectrophoresis on cellulose acetate.

Fig. 2.14 An example of the use of SDS PAGE for following the purification of an enzyme fromPseudomonas aeruginosa expressed from a plasmid inserted into E. coli (see Recombinant DNAp. 49). Lane 1, marker proteins of various Mr down to about 30 K; lane 2, total proteins expressedfrom the plasmid in the presence of the inducer IPTG; lanes 3–11, the purification of the desiredprotein by the use of an affinity chromatography column: lane 3, eluent (washing) from thecolumn; lane 4, low salt eluate, first fraction; lane 5, as for lane 4 but later fraction; lanes 6–11,proteins which were eluted from the column by imidazole; lanes 6, 7, 8, eluates from column with20 mM imidazole; lanes 9, 10, 11, eluates from column with 200 mM imidazole; lane 11, thehomogeneity of the desired protein (dimethylarginine dimethylamino hydrolase).

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electrophoresis under various conditions.To achieve this, many different methods areused, based on the characteristic propertiesof proteins, and in particular their ability tointeract specifically with small molecules.The methods used must not impair thestructure of the native protein or affect itsbiological activity.Traditional methodsinvolved the differential solubility ofproteins in solutions of ammonium sulfate,but many other methods are now available,such as gel-permeation chromatography,ion-exchange chromatography (similar tothat already described for amino acids butusing cellulose or Sephadex rather than aresin) and affinity chromatography. Someof these methods are described below.

GEL-PERMEATIONCHROMATOGRAPHY

This technique utilizes a matrix based ondextran.This cross-linked polymer ofdextran forms a mesh that can be

penetrated only by molecules of a certainsize (the greater the cross-linking, thesmaller the holes of the mesh).The tradename of the dextran is Sephadex; variousgrades of Sephadex are produced and thesediffer in the extent of cross-linking.Theprinciple of the method is shown in Fig. 2.16. Large molecules, which penetratethe mesh less readily, have less volumethrough which to permeate and thus elutemore quickly.The matrix is normallypacked in a column.The method can beused to separate small molecules, such assalts, from larger molecules, such asproteins, and to separate macromolecules ofdifferent sizes. Other materials such asagarose and Sepharose (a proprietaryagarose) can be used as the basis of thematrix.

The ability of proteins to bindspecifically to other molecules is the basisof affinity chromatography. In thistechnique ligand molecules that bind tothe protein of interest are covalently

attached to the beads in the form of acolumn.The ligands can be enzymesubstrates or antibodies.The proteins areeluted by adding an excess of the ligand or by changing the salt concentration orpH of the elutent. (See p. 78 forimmunoaffinity chromatography.)

NOMENCLATURE FOR THE SIZE ANDDENSITY OF MACROMOLECULES

Formerly, the size of a molecule wasdescribed in terms of its molecular weight,but the term relative molecular mass(abbreviation Mr) is now preferred. BothMr and molecular weight are ratios andhence it is incorrect to give them unitssuch as daltons (symbol Da). It is thusincorrect to state that ‘the Mr or themolecular weight of substance X is 105 Da’; the correct usage is ‘Mr = 10 000’.The dalton is a unit of mass equal to one-twelfth the mass of an atom of carbon-12.Hence, it is correct to say that ‘themolecular mass of X is 105 Da’ or to useexpressions such as ‘the 16 000-Dapeptide’. For entities that do not have adefinable Mr, it is correct to state, forexample, ‘the mass of a ribosome is 107 Da’.A kilodalton (symbol kDa) isequal to 1000 Da.

Gel permeation can be used for thedetermination of the Mr of a protein. Plotsof the elution volumes (Ve) of nativeproteins of known Mr on Sephadex G-75and G-100 versus log Mr are shown in Fig. 2.17.

LARGE-SCALE SEPARATION OFPROTEINS

The scheme illustrated in Fig. 2.18 showssome of the many methods that are usedfor the separation of the plasma proteins.Cryoprecipitation depends on the lessersolubility of some proteins in the cold.DEAE (diethylaminoethyl)-, QAE(quaternary aminoethyl)- and SP(sulfopropyl)-Sephadex (or Sepharose)provide separation by ion exchange, as doesCM (carboxymethyl)-Sepharose.Althoughevery effort can be taken to produce aproduct that consists only of the protein of interest, purity cannot be guaranteed.Thus the isolated protein might becontaminated with very small amounts ofother substances. Examples of suchcontamination are: (i) the virus that causesAIDS–HIV (see p. 29) in preparations offactor VIII, which is used in the treatmentof people with hemophilia;(ii) the presence of the factor that causes

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α-carbon backbone

Disulfide

Fig. 2.15 The creation of non-covalent bonds is important in the formation of the tertiarystructure of proteins. The various bonds are illustrated.

Fig. 2.16 Gel-permeationchromatography can beused for the removal ofsmall molecules fromprotein solutions and forthe separation ofproteins according totheir size.

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Creutzfeldt–Jakob disease (see p. 60) inpreparations of human growth hormone;(iii) and the virus that causes hepatitis C inproducts from blood. Some of thesecontaminants can be inactivated by heattreatment. In many cases, the alternative ofexpressing a recombinant DNA for thechosen human protein in a vector such asE. coli or yeast (see p. 49) is to bepreferred, but care must be taken toeliminate the proteins of the vector fromthe human protein preparation. Suchmethods are used for the preparation oferythropoietin, which is used for thetreatment of anemia in patients withkidney failure. Serum albumin cannot asyet be obtained in this way.

THE DETERMINATION OF THEAMINO ACID SEQUENCE OFPROTEINS

Proteins have precisely defined amino acidsequences and there are many reasons forwishing to know this sequence for eachprotein.As shown later (p. 34), it might bepossible to achieve this by an indirectmethod after determining the structure ofthe gene for the protein and deducing theamino acid sequence from knowledge ofthe genetic code. Direct methods involvethe determination of the N-terminalamino acid followed by Edmandegradation. Because this method is limitedto about 50 amino acids, it is first necessary

to break larger proteins into smallerpolypeptides, either chemically or by theuse of proteolytic enzymes (see p. 154).Provided the peptides overlap, it is possibleto deduce the sequence of the entireprotein. Edman degradation involves thereaction of phenylisothiocyanate with theN-terminal amino acid and its release bymild acid.The procedure is continued in astepwise, automated manner.

PROTEIN STRUCTURALHIERARCHIES

The polypeptide chains of proteins fold invarious ways, both within chains and withother chains.This folding is essential forthe biological activity of proteins and it isthis intricate folding that must be preservedduring the procedures involved in proteinpurification.Although it has long beenclaimed that the manner of folding of thepolypeptide chains is determined solely bythe amino acid sequence of the chains, it isnow accepted that proteins with identicalamino acid sequences can exist indifferently folded forms, and that suchfolding can be influenced by the presenceof other proteins, known as molecularchaperones (see p. 15).

It is useful to consider protein structurein terms of the four hierarchies shown inFig. 2.19.

PROTEIN DENATURATION ANDRENATURATION

A protein that possesses its own uniquebiological property is known as a nativeprotein, to distinguish it from a proteinthat has lost this property and which isdescribed as denatured.A denaturedprotein has lost its three-dimensionalstructure, also known as its conformation.Denaturation can be either irreversible orreversible.An example of irreversibledenaturation is the application of heatwhen an egg is boiled; the egg white(albumen) coagulates in an irreversiblemanner. In fact, this is a common eventduring cooking that renders proteins moresusceptible to the action of proteolyticenzymes when the food is eaten.

Reversible denaturation can beachieved by the careful use of reagentssuch as urea and mercaptoethanol. Ureadestroys the water structure and hencedecreases the hydrophobic bonding of theR-groups of the amino acid residues (seeFig. 2.15), resulting in the unfolding anddissociation of the protein molecules.Mercaptoethanol reduces the S–S bonds.

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Fig. 2.17 Gel-permeationchromatography can beused to determine the Mr ofa protein.

Fig. 2.18 Methods thatcan be used for thelarge-scale fractionationof proteins.

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It might therefore be possible to renaturethe protein when the urea andmercaptoethanol are removed.Theseprocesses are shown in Fig. 2.20 forribonuclease.

Renaturation has been taken to indicatethat a protein with the ‘correct’ primarystructure will fold spontaneously to givethe unique structure required for biologicalactivity.This process is termed ‘protein self-

assembly’. It is now realized that there aretwo means whereby renaturation can beassisted. One involves the enzyme proteindisulfide isomerase, an enzyme that plays arole ‘correcting’ wrongly paired S–S bonds.The other involves molecular chaperones,which have already been referred to.Thesecan be defined as a family of unrelatedclasses of proteins that mediate the correctassembly of other polypeptides but are not

components of the functional assembledstructures. Examples are heat-shockproteins synthesized by cells after theirexposure to an abnormally increasedtemperature.

PEPTIDES, STRUCTURE ANDBIOLOGICAL ACTIVITY

EXAMPLES OF SMALL PEPTIDES

There are many naturally-occurringpeptides with a wide range of activity, suchas hormones, first messengers inneurotransmission, local mediators andantibiotics.These peptides vary in lengthfrom the three amino acids of thyrotropin-releasing hormone (TRH) to the 231amino acids of human gonadotropin. Eventhe smallest peptides have a very specificactivity.

The structures of some typical peptidesare shown in Fig. 2.21.The N- and C-termini are often modified.Thus, inTRH, the N-terminus is a cyclizedglutamic acid (pyroglutamic acid) and thereis an amide at the C-terminus. It is possiblethat such modifications enhance metabolicstability by protecting the peptides againstexopeptidases.

Examples of small peptide hormonesproduced in the posterior pituitary areoxytocin and vasopressin.The structures of these are shown in Fig. 2.21; again, theC-terminus is an amide.The vasopressinsare more correctly named antidiuretichormone (ADH), because their mostimportant physiological action is topromote reabsorption of water from thedistal renal tubule. Oxytocin acceleratesbirth by stimulating contraction of uterinesmooth muscle.These structures illustrate

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Primary structure

Secondary structure

Tertiary structure

Quaternary structure

Fig. 2.19 Proteinstructure can bethought of in termsof four hierarchies.

UREA

Air oxidation of thesulfhydryl groups inreduced ribonuclease

Fig. 2.20 Proteindenaturation asillustrated by thetreatment ofribonuclease.

Arginine vasopressin (human)

Lysine vasopressin (pig)

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2Oxytocin

Fig. 2.21 Structures of some typical peptides.

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the specificity of peptides in that smallchanges of structure are associated withmajor functional change. Many of theantibiotic peptides are cyclized.

EXAMPLES OF LARGER HORMONES

Somatotropin (growth hormone) andprolactin are protein hormones of theanterior pituitary; lactogen is produced by the placenta.All three hormones areclosely related in structure.

Another group of hormones is a familyof glycoproteins, which includesthyrotropin, follicle-stimulating hormone(FSH) and chorionic gonadotropin.Thesecompounds all contain numerous N-linkedbranched carbohydrate chains – hence thename of the group.

Some peptide hormones are firstsynthesized as larger peptides, which aresubsequently split in the tissues into smaller peptides with discrete activities.Such large precursor peptides are called

polyproteins. Good examples are theadrenocorticotropin (ACTH) peptidesproduced from proopiocorticotropin (see p. 33).

THE USE OF THE MASSSPECTROMETER IN PROTEINSTRUCTURE STUDIES

There have been important developmentsin the approach to protein structuredetermination in recent years. In particular,techniques in mass spectrometry have beenintroduced to rapidly identify proteins andto enable the determination of polypeptideamino acid sequence. For identification,the protein is initially digested with trypsin(or similar enzyme) and the peptidesformed simultaneously analysed with veryhigh sensitivity by matrix-assisted laserdesorption mass spectrometry.The list ofpeptide masses are then compared in silico(i.e. by computer) against the computedmasses of all known proteins in the

international databases.This technique ofpeptide fingerprinting usually results inrapid identification of the original protein.

There are also approaches to obtainamino acid sequence information directlyfrom proteins but, more generally, sequenceis derived on smaller polypeptides andpeptides from a protein by collision-induced dissociation mass spectrometry. Inthis case the molecular ions of the peptidesare collisionally fragmented at theirconstituent peptide bonds and thesequence deduced from the resulting massspectrum – a process that also can becarried out automatically by computer.Thecombination of peptide fingerprinting andsequencing has led to a rapid and sustainedexpansion in studies of the proteome, inwhich complex mixtures of proteins areinitially separated by two dimensionalelectrophoresis followed by in-gel digestionand mass spectrometric analysis.

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