introduction to amino acids and proteins

8/13/2019 Introduction to Amino Acids and Proteins

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2 Introduction o amino acidsand proteins

2.1 Overview: what are proteins andwhy are they special?Proteins are biological polymers play important roles in virtually all thechemical processes of life. As such, they are abundant in all cells, repre-sentingapproximately 15 per cent of the total cell mass. There are thousandsof different types of proteins in even simple cells, but all proteins are derivedfrom the same basic building blocks: a set of amino acids. An understandingof the chemistry ofthese amino acids, and how their properties change whenthey are polymerized in a protein, is therefore of key importance in theanalysis of the molecular basis of the biological properties of proteins.

Proteins are linear polymers derived from a-amino acid monomers (seeFigs 2.1 and 2.2). These a-amino acids are carboxylic acids with an aminogroup, a hydrogen atom, and a further substituent (R) attached to thea-carbon (i.e. the carbon adjacent to the carboxyl group). In proteins, thesubstituent R is limited to one of 20 possible groups (or occasionally aderivative of one of these groups) and the individual amino acids are joinedviaamide linkagesto form a polypeptide chain. An amide linkage is the resultof the condensation of the amine and carboxylic acid functional groups ofadjacent monomers; the remaining portion of one monomer is termed anamino acid residue. A protein is typically a polypeptide chain of severalhundred such residues. Shorter polymers are frequently calledpeptides.

amide bondmino acidresidue- , ormed by

H H

Hydrolysis, i.e. +H,O Dehydration, i .e. -HO

H H 0

Fig. 2.2 Generalized protein structure: amino acids are joined in a linear chain.

,OHamino carboxyl

Fig. 2.1 Generalized structure ofan a-amino acid. In aqueoussolution these occur as zwitter-ions (see Section 2.2).

The term peptide bond is oftenused to signify the amide bondbetween 0-amino acids inpeptides and proteins.In this book, bonds designated- ndicate a continuation of thepolymer chain.

The term peptide is generallyused to signify a polymer withless than about 30 residues. Forsimplicity, in this book the termprotein is used for all biologicalpolypeptides regardless oftheir size.


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8 Introduction to amino acids and protein s

Nylon-6 is a polymerof6-arninohexanoic acid.

The importance of polar andnon-polar substituents indetermining the structures thatpolymers adopt in aqueoussolution was outlined in Section1.5.

Acidity reflects the tendency for abond to break and generate aproton. Basicity reflects thereverse process: the tendency toform a bond between a protonand an atom with an available pairof electrons. The ease with whichthese processes occur for part i-cular chemical functional groupsis quantified in terms of the equi-librium constant, Ka, for the reac-tions. A general reaction of thistype in which a functional groupHA, the conjugate acid, dis-sociates to give a proton and A - ,the conjugate base, is

H - A + H+ +A-K, is the ratio of concentrations(a more precise analysis uses'activities' rather than con-centrations) of products tostarting materials, i.e.

Proteins are not the only linear polymers of amino acids, e.g. nylon-6 is asynthetic analogue. A distinguishing feature of proteins is that they arecomposed of a variety of monomer units linked together in a definedsequence, i.e. not just a repeated pattern of one or two simple monomers asfound in a synthetic polymer such as nylon. The huge variety of possibleproteins underpins the diverse roles for which they are suited: as examples,some proteins act in a structural capacity, e.g. collagen and keratin (found intendons and hair, respectively) function in biology as fibres; others, such ashaemoglobin, are involved in transport of molecules within organisms; andyet more, enzymes, act as catalysts for the essential chemical reactions of cells.

It is the presence of a range ofdifferent functional groups (R', R", R"', etc.in Fig. 2.2 attached to the a-carbon of each amino acid residue that providesthe structural and chemical versatility essential to protein function. Thesefunctional groups branch off the polymer backbone, the main chain, and aretermed side chains. Proteins are generated in biological systems by con-trolled, sequential addition of monomer units. The specific amino acidspresent in a protein, and their order within the chain, give rise to the dis-tinctive shape and chemical functionality of each final polymer.

In order to understand the structures and functions of proteins, thechemistry of the functional groups present in the protein will be examined.Proteins usually adopt highly ordered structures in the cellular environment,based on the nature of the substituents on the polymer chain (polar and non-polar) and on the stereochemical features of both the amino acids and thepeptide bonds that link them. An appreciation of these structural principleswill be used in analysing case studies of well-studied proteins in later chapters.

2.2 The acid base chemistry of a aminoacids and proteinsAn understanding of the structures and simple chemical features ofa-amino acids is important for appreciating the properties of proteins.Acidity and basicity of amino acids, and hence of proteins, is the chemistrywhich will be discussed in most detail. These are important chemicalproperties in biological systems. Simple acid-base chemistry will be illu-strated using glycine, the simplest amino acid. Initially, the properties offree glycine will be described, followed by a discussion of the way in whichthese properties are modified when glycine is incorporated into proteins.2 2 1 GlycineThe simplest amino acid is glycine, in which two hydrogens are attached tothe a-carbon, i.e. the substituent, R in Fig. 2.1, is hydrogen. The overallchemical properties of such a molecule reflect the combination of theproperties of the various functional groups of the molecule, each of whichhas a distinctive chemistry.

.&OHGlycine exists primarily as chargedspecies in aqueous solution asexplained later (see Fig. 2.5). 0Glycine


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Foundations o chemical biology 9In glycine, the only functional groups attached to the hydrocarbon

framework are an amino group and a carboxyl group. Each of thesegroups can exist in different protonated forms, depending on the pH.

In strong acid solution the carboxyl group will be present in the freeacid form (i.e. as a C 2H group). This group is acidic as it can lose aproton, in response to the addition of base, to form a carboxylate ion.

H+ Rvo-RKoH=== 0The change of pH as base is added to an aqueous solution of a typical

carboxylic acid is shown in Fig. 2.3. At pH values above ca 5 (the pK ofthe acid), the carboxylate form predominates. In this case, the conjugatebase is a charged species, and the acidity of the carboxylic acid groupreflects the stability of this anionic species relative to the unchargedmolecule in aqueous solution.

The characteristic features of a base are the presence of a non-bondedelectron pair and a relatively high stability of the corresponding proto-nated form. The other functional group of glycine is an amine whichcontains a nitrogen atom with a non-bonded pair of electrons.

Mid-point(pH = pK,)-----

Ka= [RCopl[H lRCO,H] IAt the mid-pointTherefore Ka= [H][RCOz-] = [RCOZH]

A M \_ _ _ _ Equivalencewhen the amountointof base added isequal to the original- -- - - - - -

I amount of acid)Predominant I Ispecies :

Amount of base addedRCO2H I

B

Fig. 2.3 Titration curve for a typical carboxylic acid.

0 Amount of base addedFig. 2.4 Titration curve for a typical amine.

Theacidityof carboxylicacids isduetothestabilityofthe carbox-ylate anion relative to theacid.Thecharge of the carboxylate resides,primarily, on two equivalent elec-tronegative oxygens; each oxygenhas o accommodate only half anegative charge.The equivalence of the two oxy-gens in the carboxylate anion isnot clear in a single structurecomprised of simple bonds andcharges. This problem can beaddressed by imagining the over-all structure as a weighted aver-age of simple structures (canonicalor resonance orms). Here twostructures are drawn, corre-sponding to the charge residing oneither of the two oxygens. Thesestructures are equivalent and haveidentical energies. The real struc-ture is envisaged as the averageof these extreme representationsnota rapidly interconverting mix-

ture of two discrete structures).

The relationship between thesetwo canonical forms can beshown using curly arrows whichare used in organic chemistry todenote the movement of pairs ofelectrons.

R~ oo5The negative00.5 chargels shared.

Acidity is quantified in erms of pKavalues. At the mid-point of a titra-tion, an acid, HA, is half deproto-nated, the concentrations of HAand A- are equal, and the equili-brium constant K, = [H+]. Theequilibrium constants for differ-ent acid-base reactions havewidely differing values. By ana-logy to pH, logarithms are usedto compress the scale. Sincemost acids of interest are onlypartly dissociated, the sign of thelogarithm is changed sothat most values are positive


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10 Introduction to amino acids and proteinsnumbers:

i.e. log K = pK,The smaller the value of the pK,,the stronger the acid.Some approximate pK, values:Alcohols, ROH, 17Carboxylic acids, RC02H,4-5Thiols, RSH, 8Ammonium ions, RNH 10.A zwitterion is a neutral moleculethat carries equal numbers ofoppositely charged functionalgroups. Zwitterions have many ofthe properties of salts.pK, values vary somewhat,depending on the precise mole-cular structure and the environ-ment in which the acid-basechemistry is taking place.For example, the carboxylic acidgroup of glycine is more acidicthan a simple carboxylic acid(pK, = 2.35 rather than ca 4-5since the corresponding carbox-ylate anion is more stable in thepresence of the adjacent, posi-tively charged, ammonium ion.The effect of the environment onpK, is particularly important innon-polar conditions, such as inthe interior of proteins.[R < r H L R * H R ] 0

Canonical structures ra bonding andcharge distributionFig. 2.6 The structure andbonding of amides.

When a nitrogen is positionedadjacent to an unsaturatedcarbon in a molecule, it is gen-erally found to adopt a trigonalplanar, rather than pyramidal,shape. By adopting this shape,the C-N bond is more stable: theelectrons are delocalized in apartial double bond.

predominates I

Fig. 2.5 Titration curve for glycine.

The conjugate acid of an amine, a substituted ammonium ion, is rela-tively stable in aqueous solution. Such an ion in aqueous solution can bedeprotonated, but less readily than a carboxylic acid; the pK, is corre-spondingly higher. The acid-base equilibrium and titration curve is shownin Fig. 2.4. Only at a pH above ca 10 (the pK, of the substituted ammo-nium ion) does the amine exist predominantly in the uncharged form.

Since glycine contains both an amine and a carboxylic acid moiety, itstitration behaviour incorporates both features, as can be seen in Fig. 2.5.The pK, values of the carboxylic acid and ammonium groups of glycineare 2.35 and 9.78, respectively. In neutral aqueous solution (pH 7) boththe functional groups of glycine are charged (Fig. 2.5). Therefore, glycineand other amino acids are really ammonium carboxylate zwitterionsrather than amino acids; subsequent structures will reflect this.

2 2 2 The chemistry of amides: polymers of glycineWhat are the acid-base properties of glycine when incorporated into apolypeptide? When the simple dimer, glycyl-glycine, is compared with itsconstituent monomers, the ammonium group of one glycine and the car-boxylate group of the other are still present in the dimer, and their acid-base chemistry is little affected. The other ammonium and carboxylategroups are no longer present, since they are linked to form an amide bond.

Amide bond-H 0O- H3N+ 0

0 H O HGlycyl-glycineBy contrast with the nitrogen atom of the amine, the amide nitrogen is

essentially non-basic. There is an additional interaction between thenitrogen and the unsaturated carbon atom, resulting in partial double-bond character. This can be depicted using canonical structures (Fig. 2.6),in an analogous fashion to those drawn to explain the special stability ofthe carboxylate anion.


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12 Introduction t ami no acids and protein sBecause of the carboxylic acidfunction, aspartic and glutamicacids are often termed acidicresidues. This terminology can beconfusing: since they are usuallyfound in proteins in the conjugatebase form, the side chainsof theseamino acids often act as bases inbiochemistry (see Chapter 5 .For amino acid side chains whichare involved in acid-base equili-bria, the acid or base form whichpredominates at physiological pHis boxed in the structures whichaccompany this section of thetext.The titration curves for asparticand glutamic acid residuesresemble those of simple car-boxylic acids (cf. Fig. 2.3). Notethat the exact pK, of functionalgroups varies a little dependingon the environment (either inresponse to other functionalgroups in the molecule, or in thesurrounding solvent, etc).

The amino acids in this sectionare often said to have basic sidechains.This reflects the fact thatthey are readily protonated. If theyare present in a protein in anunprotonated form, they can actas bases. If they are present in aprotonated form, they can act asacids.

2.3 .2 Amino acid residues with carbox ylic acid side chainsThe side chains of two of the amino acids, aspartic and glutamic acids,contain carboxylic acid functional groups linked by a hydrocarbon spacer,of one or two methylene groups respectively, to the a-carbon. The sidechain of each of these groups behaves as a simple carboxylic acid with apK, of approximately 4 5.At neutral pH, these groups will, therefore, bepresent in the anionic conjugate base form (aspartate and glutamate).

H H 0 0 0 0H H 0 0 0 0aspartate residue glutamate residue

2.3 .3 Amino acid residues with amide side chainsA further two amino acids, asparagine and glutamine, are closely relatedto aspartic and glutamic acids. In these, instead of a carboxylic acid, theside chain contains an amide group. Amides can participate in hydrogenbonding, but they are neither strong acids nor bases, and do not affect theacid-base chemistry of proteins.

f CoNH2 r C O N H 2 N Y

glutamine residue N YH H asparagine residue

02.3.4 Acyc lic amino acid residues with basicnitrogen-containing side chainsTwo of the protein amino acids have side chains consisting of a linearcarbon chain terminating in a basic nitrogen functional group. The sidechain of lysine is a four-carbon chain ending in an amino group. This pri-mary amine bearsa non-bonding electron pair and isof similar basicity to theamines considered previously. The pK, of the corresponding ammoniumion is 10.5and, at neutral pH, this group is present in solution as a cation.

0 lysine residue

H +(xH H 0 arginine residue

In the case of arginine, protonation of the basic nitrogen leads to acation in which the positive charge is dispersed over three nitrogen atoms.This factor ensures an enhanced stability to the protonated form ofarginine which has a pK, of 12.5 and is present as a cation under phy-siological conditions.

It is a useful exercise to drawcanonical structures of theprotonated form of arginine toillustrate the dispersal ofchargeover all three side chain nitrogens.


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Foundutioiis of chemical biology 132.3.5 Amino acid residues with hydroxyl functional groupsThe side chains of three amino acids contain hydroxyl groups. Serine andthreonine are simple alcohols. For each of these residues, the hydroxylgroup is attached to a carbon adjacent to the a-carbon. Threonine is \ qR 0distinguished from serine by an extra methyl group that makes it a sec-ondary, rather than a primary, alcohol. An isolated hydroxyl group canact as an acid or a base, but neither process is especially favourable (thepK, of the hydroxyl of serine is approximately 16).In tyrosine, the hydroxyl function is attached to an aromatic ring. Herethe functional group is a phenol. The aromatic ring stabilizes the chargeon the deprotonated form. This enhances the stability of the conjugatebase and lowers the pK, (to ca 10) facilitating acid-base chemistry. Tyr-osine is usually found in the hydroxyl form, but it is occasionally found toact as an acid under physiological conditions.

serine residue

H3C OH

N ZH HI;Tthreonine residue

0

tyrosineH H

0residue

.o

2.3 .6 Sulphur-containing amino acid residuesThe side chains of two protein amino acids have sulphur-based functionalgroups. Cysteine is the sulphur analogue of serine, containing a thiolfunctional group rather than a hydroxyl function. In aqueous solutionsuch groups are moderately acidic (pK, ca 8). However, the properties ofsulphur differ from those of oxygen and thiols do not form stronghydrogen bonds. In general, sulphur-containing side chains behave asrelatively non-polar groups. In addition, the thiol group has unique che-mical properties: it is the most readily oxidized of all the functional groupsunder consideration (Fig. 2.7). When two thiols are oxidized, a disulphidebond results. Disulphide bonds are important features of some proteinstructures and are considered in Chapter 3.

CSHcysteine residue methionine residue

The amino acid methionine contains a thioether group rather than athiol. For the present discussion, the most significant feature of themethionine side chain is its generally non-polar character.

The negative charge of thedeprotonated form of tyrosine canbe dispersed over three of thecarbons of the aromatic ring.Convince yourself about thecharge distribution of theconjugate base of tyrosine bydrawing canonical structures.

PH s\

NAf\Nf.+H0 0

Disulphide-linked residues,known as a cystine residue

Fig. 2.7 Redox chemistry ofcysteine.


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14 In roduction t amino acids and proteinsA heterocyclic compoundcontains a r ing in which one of thering atoms is not carbon.

H.*ryptophan residueH H 0If both protonated and non-protonated forms are present,then the side chain can act asboth an acid and a base. This isthe situation for histidine. For thisreason, histidine residues areoften important in catalysingbiochemical acid-baseprocesses. The special chemistryof histidine is important in thefunctioning of many proteins.Twoof the succeeding chaptersdescribe proteins which haveessential histidine residues: theglobins in Chapter 4, andtriose phosphate isomerase inChapter 5.

0The side chain links back andconnects to the a-nitrogen, forminga ring.

Tetrahedral arrangement of foursingle bonds to carbon:

2.3.7 Amino acids containing nitrogen heterocyclesThe final three protein amino acids are rather different from one another,but they each contain cyclic structures involving nitrogen which areresponsible for their distinctive chemistry.

Tryptophan has a nitrogen embedded in a large aromatic framework(an indole) which behaves as a non-basic nitrogen, although it can formhydrogen bonds. Tryptophan is more like the side chain of phenylalaninethan most of the remaining nitrogen-containing side chains. It is ahydrophobic residue.

Histidine also has a side chain with an aromatic ring. In this case thering (an imidazole) has two nitrogen atoms, and can be protonated. Thecharge on the cation of the protonated form is dispersed over the twonitrogen atoms. Histidine is moderately basic with the pK , of the con-jugate acid being ca 7. Such a pK , allows both conjugate acid and baseforms to be readily accessible at neutral pH. Histidine is ideally placed toact as an acid-base catalyst in proteins operating at around pH 7.

Protonated and neutral H Hforms of histidine are bothphysiologically mportant.This evenly balancedhistidine's key role inbiological acid-base H H H H

histidine residue

equilibrium underpins H

chemistry. 0 0

Finally, proline is fundamentally different from the other protein aminoacids. The side chain comprises a three-carbon chain and, as with otherhydrocarbon side chains, contributes no unusual chemical features to theamino acid or derivatives. In this case, however, a ring structure is formedby the side chain linking back to the a-amino nitrogen. This cyclicstructure constrains the shapes which this amino acid can adopt. As aresult, the presence of proline in a protein has significant effects on itsthree-dimensional structure (see Section 3.3).

2.4 The stereochemistry of a amino acidsStereochemistry is concerned with the shape of molecules. The propertiesof molecules are strongly influenced by their stereochemistry. An under-standing of the stereochemical features of amino acids is essential for anappreciation of the fundamental principles of the structures of proteins.All amino acids except glycine have four different substituents attachedto the a-carbon. There are two distinct ways in which these substituentscan be arranged in three-dimensional space. These two forms, con3gura-tions, are mirror images of each other. All amino acid residues in naturalproteins have the same absolute configuration. These properties are illu-strated in Fig. 2.8 for a typical amino acid, serine.

In serine the a-carbon is surrounded by four different groups: anammonium, a carboxylate, a hydroxymethyl and a hydrogen atom. The


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16 Introduction to ainino acids and proteinsAn abbreviated version of theC-I-P rules is given below(i) PrioritiesDecreasing priorities, numbered1-4, are assigned to the atomsattached to the chiral centre.Higher priority is given to atomsof higher atomic number. In thecase of all chiral amino acidsfound in natural proteins, thisleads to the pr iorities N >C(carboxyl), C (side chain) > H.Theamino group is numbered 1 andthe hydrogen atom 4. When, ashere, consideration of theseatoms leaves an ambiguity, it isresolved by extending the rules tothe next attached atoms. Thepriority of these atoms isassigned, as before.(ii) Translating priori ties intodescriptionsThe chiral centre is viewed fromthe side opposite the lowest-prior ity ligand. If the direction ofdecreasing priority (1,2,3) of theother three groups is clockwise,the configuration is designated R,whereas an anticlockwise order-ing is designated s.

It is a useful exercise to draw outthe structure of cysteine; assignpriorities to the substituentsaround the a-carbon according tothe C-I-P rules; and use thesepriorities to assign theconfiguration of i-cysteine as R.

The chemists view:assigning R and SThessigningiochemistsand Dview: //// / +CH3

View from the side of from the side co;cO, the hydrogen atom

3N 0 C H 3Identify the carbonyl (CO) of the carboxylgroup, the side chain substituent R, in thiscase CH,) and the amino (N) groups

N&For L-amino acids,following the substituentsin a clockwise mannerspells CORNAssign C-I-Ppriorities to the threenon-hydrogen groups

2

3aecreasingpriorities runninganticlockwiseindicates SFig. 2.9 Identifying the configuration of amino acids: alanine as an example.

The C-I-P rules involve assigning priorities to the groups attached tothe chiral centre and then relating these priorities to a description of thechiral centre. The two possibilities are known as R and S configurations.As an example, these rules are illustrated for alanine in Fig. 2.9.

In the case of alanine, the priorities of the atoms attached to thea-carbon are N > C >H. Hence the amino group is numbered 1 and thehydrogen 4. Of the two carbon substituents, the side chain carbon isbonded to hydrogen, whereas the carboxyl carbon is bonded to oxygen;hence the latter has the higher priority. The overall priority order is,therefore, N >C (carboxyl) > C (side chain) > H. When the a-carbon isviewed from the side opposite the hydrogen (priority 4), the decreasingpriority of the other groups follows an anticlockwise pattern, and theconfiguration is designated S.

All chiral amino acids found in natural proteins have the S configura-tion, except cysteine where the presence of a sulphur atom only onecarbon removed from the chiral centre changes the priority rules about thea-carbon. Thus although cysteine has essentially the same shape as serine,it ends up with the opposite stereochemical descriptor This kindof anomaly does not deter organic chemists who can see the benefitsof the C-I-P rules in a wider context, but is sufficient to make manybiochemists prefer the original Fischer terminology. Hence the biologicalchemistry literature includes both notations and students should befamiliar with both.


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18 In t roduc t ion t o amin o ac ids and pro te insIrrespective of whether a peptidebond to proline is cis or t rans thea-carbon of the other amino acidis involved in unfavourable stericinteractions with a carbon sub-stituent attached to the prolinenitrogen.In natural proteins about 10percent of all peptide bonds involvingthe nitrogen of proline are cis.This poses particular problemsfor generating unique structuresfor proteins with prolines.

Greater steric clash

Steric clash similarto that of a 'normal'cis-peptide bond andcomparable to thatof the trans-peptide Ibond to proline , ; 0

In the case of peptides involving the nitrogen of proline, this preferenceis smaller since the a-carbon of the neighbouring residue must avoid eitherthe a-carbon or the other carbon attached to the nitrogen; hence the isform is not dramatically disadvantaged relative to the t rans form.

2.7 SummaryProteins are linear polymers of a-amino acids linked via peptide bonds.Each of the 20 monomers bears a characteristic side chain that introduceschemical diversity into the polymer. Side chain substituents include arange of non-polar and polar groups. An appreciation of the acid-baseproperties of the functional groups found in these side chains is essentialto a proper understanding of protein chemistry. The absolute orientationin space of the amino, carboxyl, hydrogen and side chain is the same for allprotein amino acids and is usually referred to as the L-configuration,although the R nomenclature of organic chemistry is also widely used.The peptide bonds that link monomeric amino acids are planar andgenerally exist in a t rans rather than a is form. Proline, with its cyclicstructure, and glycine, with two hydrogens on the a-carbon, have unusualstructural features that set them apart from the remainder of the aminoacids.

Further readingM . Hornby and J . Peach (1993) Foundations of Organic Chemistry, OxfordChemistry Primer, Oxford University Press, Oxford, provides a useful intro-duction to organic chemical conventions.J. Clayden, N. Greeves, S. Warren and P. Wothers 2000) Organic Chemistry,Oxford University Press, Oxford, and C. K. Mathews, K . E. van Holde andK. G. Ahern (2000) Biochenzistrj., 3rd edn, Benjamin/Cummings, San

Francisco, are excellent general textbooks for organic chemistry and bio-chemistry, respectively.For information on other aspects of amino acid and protein chemistry, see:J. H. Jones (1992)Amino Acid and Peptide Synthesis, Oxford Chemistry Primer,Oxford University Press, Oxford.

introduction to amino acids and proteins

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