Protein Structure PDF

10 Biomolecules and Catalysis Standard amino acids with Nonpolar side chain Polar side chain Glycine (Lowest MW) Tryptophan (Hi...

10 Biomolecules and Catalysis Standard amino acids with Nonpolar side chain Polar side chain Glycine (Lowest MW) Tryptophan (Highest MW) Alanine Uncharged at pH 7 Charged at pH 7 Valine Serine Leucine Threonine Isoleucine Cysteine Proline Negative charge Positive charge Asparagine Methionine Glutamate Lysine Glutamine Phenylalanine Aspartate (Most acidic) Histidine Tyrosine Arginine (Most basic) Selenocysteine (Sec or U)is the 21 standard amino acid. It has astructure similar to that of cysteine, but it contains selenium rather than sulphur. It is incorporated into polypeptides during translation. However, it is specified by a triplet codon, UGA (a stop codon). Selenocysteine has its own tRNA containing the anticodon UCA and it is formed by modifying a serine that has been attached to the selenocysteine tRNA, Enzymes like glutathione peroxidase and formate dehydrogenase contain selenocysteine in their catalytic center. Pyrrolysine (Pyl or O) is the 22n standard amino acid. It is similar to lysine and is present in some bacterial proteins. It is coded by UAG codon. COO H,N CH, CH, CH, COO COO CH, H,N-C-H H,N-CH NH CH, CH, C=0 Se H,C, H H Cysteine Selenocysteine Pyrrolysine Problem Determine whether the following statements are true or false. If false, explain why? 1. All 20 standard amino acids found in proteins have at least one asymmetric carbon atom. 2.3. AnAlanine equimolar mixture of D- and L-aianine does not rotate the plane of polarized light. obtained from a protein hydroiysate has the same absolute configuration as D-glyceraldehyde. Solution: 1. False: glycine has no asymmetric carbon atom. 2 True. L-glyceraldehyde. 3. FaBse: alanine obtained from a protein hydrolysis has the same absolute configuration as  Biomolecules and Catalysis 1.1.4 Titration of amino acids Because amino acids contain ionizable groups, the predominant ionic form of these molecules in solution depends on the pH. Titration of an amino acid illustrates the effect of pH on the net charge of amino acid. It involves the gradual removal of protons with increasing pH. Let us understand the titration of non-ionizable R group containing and ionizable R group containing amino acids separately. Standard amino acids with Non-ionizable R-group Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, Ser, Thr, Asn and Gin Ionizable R-group Asp, Glu, His, Arg, Tyr, Cys and Lys Amino acid with ionizable R-group Amino acid with non-ionizable R-group Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, Ser, Thr, Asn and Gin Asp, Glu, His, Arg, Tyr, Cys and Lys pk, = pk, pK, pk, cOOH COOH H,NCH H,N-CH pK, - pk2 CH, pK, pk, pk, pk, CH,R R COOH Titration of non-ionizable R qroup containing amino acids and alanine) have two ionizable groups (a-amino and Non-ionizable R groups containing amino acids (such as glycine Let us understand the titration of alanine. During a-carboxyl group) that can undergo protonation and deprotonation. protons in a stepwise fashion. Ata very low pH (i.e. titration with a strong base such as NaOH, alanine loses two alanine is the fuly protonated form in which the a-carboxyl strongly acidic solution), the predominant ionic species of charge on the molecule is charged. Under this condition, the net group is uncharged and a-amino group is positively (-NH) the deprotonation of a-carboxy! (-C00H) and a-amino +1 (Ala). However, an increase in the pH results in loses its proton. After deprotonation of a-carboxyl group, groups. In the first stage of the titration, the -COOH group Its -COOH group has a pk, (labeled pk,) of 2.34. the net charge on the molecule becomes zero (Ala"). 1 7 pH range: -1 Net charge: +1 COO Coo COOH DK, 2.3 DK,9.) H,N-CH H,N C,H H,N CH, CH, CH, Ala Ala Ala Fully protonated Zwitterion Fuliy deprotonated raised, the nost Figure 1.6 At very low pt, alanine is fully protonated, and Ala" is the predominant species present. As the pH is of Aig' and Ala are equai proton, which has the iower pk,, The fractions acidic proton dissociates; this is the carboNyic acid protonated a-amino group. hich second dissociation occurs, This is the when pH pk, 2.3. As the ptt is raised further, the As the pH is raised furthe, As beconmes the are equat with pH pk, x 9,7. has the higher pK. The fractions of Aia and Ala only species present,  Bomolecules and Catalysis the titration of alanine proceeds, a is an equal concentration of Ala and Ala". As point 0S reached t pk,, there complete and removal of the seCond of the first proton is essentially has just pH nis 6.01, pH, at alanine which is removal present largely as the dipolar ion (zwitterion) i.e., alanine has no net charge and is begun. electricallyAt isoelectric point (pI). Because there is no net this occurs is called the eutral (Ala"). The pH at which electrophoretically non-mobile and least soluble at this pH. charge at the oelectric point, an amino acid is Further increase in group has a pK, (labeled n k e e in group. lts -NH3 Hresults in the deprotonation of the charged a-amino Ala-1, At high pH (about 12), alanine is completely deprotonated concentration of Ala' and K2, there is an equal deprotonated and a net pe pd the net charge on the molecule is -1 (Ala ). Ihe amino group lS Completely of the carboxyl group. harge develops due to the presence 13 Figure 1.7 Alanine Titration of alanine. A plot Ala = Ala-1 of the pH of the solution versus the amount of OH added yields pk, = 9.7 a titration curve. 1 equivalent pk,= 2.3 of OH = 0.1 M NaOH added. At pH 1.0, for example, alanine COOH 7 exists almost entirely as the form pH pl = 6 Alaa with anet positive charge of 1. H,NCH At pH 2.3, where there is an equal mixture of Ala +1 and Ala, the pk,= 9.7 Ala= Ala average or net positive charge CH, is +1/2. At pH 9.7, where there is pk, = 2.34 an equal mixture of Ala and Ala, the average or net negative charge is -1/2. At pH 12, alanine exists almost entirely as the form 0.5 1 1.5 2 Ala-1 with a net negative charge. Equivalents of OH added charge. The At pH 6, alanine is present predominantly as its dipolar form, fully ionized but with no net electric Its characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH (pI). isoelectric point is simply the arithmetic mean of the two pk, values: pI pk, + pK2 2.34 + 9.7 =6.0 2 2 Ala+ than in form Ala; in this At apH value lower (more acidic) than the pI, more alanine molecues are in form than the pl, more alanne Situdtol, alanine molecules has anet positive charge. At a pH value greater (more basic) molecues are in form Ala' than in form Ala9, In this situation, alanine molecules has a net negatve cnerg it is possible ro a NOte: AIthough at any instant an individual amino acid molecule will have an integral charge, Popuiation of amino acid molecuies in solution to have a fractional charge. For example, at pH 1.0 dlame equal mixture of entirely as the form Ala with a net positive charge of 1. However, at pH 2.34, where there is an +1 Ala and Ala,the average or net charge on the population of alanine molecules is 0.3. 2.3 pH less than Present predominantly as Ala* 2.3 |pk, = 2.3 pH equal to Average net charge is +1/2 9.7 COOH Half of the amino groups are ionized pH equal to 2.3 pH equal to H,N Ca pH is equal to the pk, of the carboxyl group equal to 9.6 -H pH is equal to the pK, of the protonated amino group pH to pl pk,=9.7 CH, pH equal Predominant species is Ala" pH equal2.3 Alanine Present as a 50:50 mixture of Ala and Ala" to 9.7 Average net charge is -1/2 pH equal  Biomolecules and Catalysis 13 Titration of ionizable R group containing amino acids a-amino acids with an ionizable Rgroup (such as glutamic acid, histidine and lysine) have three possible deprotonation steps; thus they have three pk, values (pKj, pkg and pk,). Let us understand the titration of glutamic acid. It has an a-C00, an a-NHj and a y-CO0 group in the side chain. The pk, of a-COOH, a-NH; and y-COOH are pki, pKz and pka, respectively. At very low pH, the glutamic acid has a net positive charge of +1 (Glu*) because a-NHj group has apositive charge. During titration, as strong base such as NaOH is added and the pH increases, the a-cOOH group loses its proton to become negatively charged, and the glutamic acid now has no net charge (Glu"). As still more base is added, the y-COOH group present in side chain loses its proton, and this is the pH at which the glutamic acid has net negative charge of -1 (Glu'). At still higher values of pH, the a-NHj group loses its proton and the glutamic acid now has a negative charge of -2 (Glu). COOH coo" cOo COo" pk, 2.19 pk = 4.25 pk, = 9.67 H,N-C-H H,NC-H H,N-C-H H,N-C-H CH, CH, CH2 CH, CH, CH, CH, CH, COOH COOH COO COO Gu Glu Glu Glu 14 Glutamic acid 12 + |DK, =2.19 10+ pk, COOH 8 - pH H,NC-H 6 PK,=9.67| CH, pKR CH, pK, COOH 2 pk=4.25 1.0 2.0 3.0 Equivalents of OH added Fiqure 1.8 Titration of glutamic acid. At very low pH, the glutamic acid has a net positive charge of +1 because a-amino group has a pOsitive charge. As the base is added (pH increases), the a-carboxyi group loses its proton. Glutamate now has no net charge. As still more base is added, the y-carboxyl group loses a proton, and the molecule has a -1 charge. Adding base further results in loss of protons from ammonium ion. At this point, glutamate has a net charge of -2. The isoelectric point of glutamate is simply the arithmetic mean of the two pk, values: pk, and pke. It has a pI of 3.22, considerably lower than that of alanine. This is due to the presence of two carboxyt groups. The pl value for glutamate is the pH halfway between the pK, values for the two carboxyl groups: pk, pkg 2.19 4.25 p! 3.22 2 Histidine alsohas three ionizable functional groups, thus has three pk, values (pk, = 1.82, pKg = 6.0and pk, = 9.17). At very low pH, the histidine molecule has a net positive charge of +2 because both the imidazole and a-amino  Biomolecules and Catalysis 14 added and the pH increases, the a-carboxyl groups have positive charges (His*). As base is group oses proton its now has a positive charge of +1 (Hist). As still more to become negatively charged, and the histidine is the point at which the histidine has no net base is added. proton, and this the charged imidazole group loses its proton and the histidine molecule now has a charge. At still higher values of pH, the a-amino group loses imidazole its group negative charge can be readily ionized near the physiological pH. Consequently, of near 6, the fos..dently, -1. With a pke value at pnysiological pH. Histidine is often can be uncharged or positively charged imidazole group release protons in the course of enzymatic enzymes, where the imidazole ring can accept or reactions. Due sites of neutral nu only histidine provides significant buffering power near the to pkp Value near 6, COO COO COOH COO H,NC -H H,N-C-H H,N-C-H H,NC-H pK, = 1.82 pkg = 6.00 CH, pK, = 9.17 CH, CH, CH, HN -NH -NH NH +2 His His His His of 2 because both the imidaole Figure 1.9 Titraton of histidine. At very low ph, the histidine moBecule has a net positive charge a proton to become and a-amino groups have positive charges. As base is added and the pH increases, the a-carboxyl group loses charge of +1. As still more base is added, the charged imidazole oro. negatively charged, and the histidine now has a positive loses its proton, and this is the poit at which the histidine has no net charge. At still higher values of pH, the a-amino aroup loses its proton and the histidine molecule now has a negative charge of -1. Problem An amino acd contains no 1onizabie group in its side chain (R). It is titrated from pH Oto 14. Which of the following state is not observed during the entire titration in the pH range 0-14? R R A. H,N-CH--coo 8. H,N CH-cOOH C. H,N--CH -COo D. H,N-CHcOOH Solution: The state of amino acid mentioned in option Dwil not observe during the entire titration in the pH range 0-14? Problem An amino acid has one protor donating carboxyl group in the side chain. The pK,, pk, and pke values for this ano are 2.19, 9.67 and 4.25, respectiveiy. What will be the predominant ionization state of amino acid at pH 3, 7 an0 L4 Solution: The net charge on an arnno acid depends on pH. At a pH of 1. the a-Carboxv aroup, the carboxyl group side chain, and the a-arnano group wit remain protonated. Hence, the net charge on amino acids will be -l. nowe. pH of 3, the e-cartboxy group witt Sose a proton, but the remaining aroups will renain orotonated, Thus, a net charge zero. At a pH of 7, the -carboxy group and the carboxyi group of the side chain will lose protons, and the u-amino group will remain protanated. Hence, the ret charge on amino acids will be -1. At a pH of 12, all functional groups willlose protons Hence, the net charge on amino acds wti be -2 At pH 1 At pt 3 At pH 7 At pH 12 COOH COo CO0 H,N-C --H H H,N--C-H R-COOH COO R-CO0 +1 Net charge 0 -2  Biomolecules and Catalysis 15 Table 1.2 pk, values for the ionizing groups of the standard amino acids Amino acid MW pK, (-cooH) pK; (-NH$) pkR pI Non-polar side chain Glycine Gly, G 75 2.34 9.60 5.97 Alanine Ala, A 89 2.34 9.69 6.01 Valine Val, V 117 2.32 9.62 5.97 Leucine Leu, L 131 2.36 9.60 5.98 Isoleucine Ile, I 131 2.36 9.68 6.02 Proline Pro, P 115 1.99 10.96 6.48 Methionine Met, M 149 2.28 9.21 5.74 Phenylalanine Phe, F 165 1.83 9.13 5.48 Tryptophan Trp, W 204 2.38 9.39 5.89 Polar and uncharged side chain at pH 7 Serine Ser, S 105 2.21 9.15 5.68 Threonine Thr, T 119 2.11 9.62 5.87 Asparagine Asn, N 132 2.02 8.80 5.41 Glutamine Gln, Q 146 2.17 9.13 5.65 Cysteine Cys, C 121 1.96 10.28 8.18 (suifhydryl) 5.07 Tyrosine Tyr, Y 181 2.20 9.11 10.07 (phenol) 5.66 Polar and charged side chain at pH 7 Positively charged Histidine His, H 155 1.82 9.17 6.00 (imidazole) 7.59 Lysine Lys, K 146 2.18 8.95 10.53 (e-NH) 9.74 Arginine Arg, R 174 2.17 9.04 12.48 (quanidino) 10.76 Negatively charged Aspartic acid Asp, D 133 1.88 9.60 3.65 (B-COOH) 2.77 Glutamic acid Glu, E 147 2.19 9.67 4.25 (COOH) 3.22 Note: pk, values depend on termperature, ionic strength and the microenvironment of the lonizable group Seven am.n0 2c1d side ch¡ins contaio aroups that ionize between pH 1 and 14. For Asp, Glu, fyr and Cys, the ionizable groups are uncharged below ther p ard neg atively charged above thelr pka- For His, Lys and Arg, the ionizabie groups are positively charged below ther p, and uncharged above their pKR All a-amino acids have pk, of the -COOH group in the range of 1.8 to 2.4, and p, of theNH; group in the range of 8.8 to 11. Amino acids with high isoelectric points are classified as basic amino 3CIds. Lvsine and argin1ne are the two most basic amino acids. The amino acids aspartic acid and glutamic acid have low iSoelectric points. Amino acids with low isoelectric points are classified as acidic amino acids  Bioonclecules and Cata)sis ionizable side chains at different pH standard amino acids with able 1.3 lonic states of At pH 7 At low phH At high ph -C0OH --CO0 -C00 Aspartic acid 3.65 -C00H -C00 -CO0 Gutanic aod 4.25 -SH -S 8.18 -SH Csteine -NH; -NH, Lysine 10.53 -NH; Histidine 6.00 Arginine 12.48 NH; NH Tyrosine 20.07 Nicte: Bot natra and protonatet forms of hstine are present t pH 7, since ts pk is close to 7. Absorption of UV radiation by aromatic amino acids Argmatic amno acds such as tryptophan, tyrosine and phenylalanine absorb utraviolet (UV) light. The aromatic side chains of these amino acids are responsible for W absorption. Phenylalanine and tyrosine are aromatic due to the presence of a pheny group and a4-hydroxy pheny group, respectively. Tryptophan is aromatic due to its heterocyclic indole rng Tryptoghan and tyrcsine absorb maximum near a wavelength of 280 nm. However, phenylalanine absorbs maxm t 257.4 om. Absorbance at 280 nm is used for detection and quantification of purified proteins. Proteins containing tyrosine and tryptophan absorb W radiation at a wavelength of 280 nm. Phenylalanine also contributes in the absorption at thes wavelength but since t is relatively insigniñcant, it can only be observed in the absence of both tryptuphan and tyrosine. Proteins of similar molecuiar weight can have different absorbance values due to the arterence in trvyptoghan and tyrosine content. UW absorbance is also affected by protein structure. Thus, corndtions which afect structure (such as temperature, pH, ionic strength or the presence of detergents) can affect the abity of aromatic residues to atsorb ight at 280 nm. Trp Absorbnce Absorbance of amino acids at neutral pH Amino acids Absorbance (ma) Tyr Phenyiaianine (Phe) 257.4 nm Tyrosine (Tyr) 274.6 nm Phe Tryptophan (Trp) 279.8 nm 230 240 250 260 270 280 290 300 310 Wavelength (nm) Figure 1.10 Absorption of UV-lhght by the amino acids are present in equimolar amounts (10 aronmatic amino acids tryptophan, phenylalanine and tyrosine, at pH 6.0. The M) occurnear wavetength of 280 nm and absorbance of Trp is under sdentical conditions Maximum absorption for both Trp and Tyr as much as four tines that of Tyr.  Biomolecules and Catalysis 17 1.1.5 Peptide and polypeptide covalentiy linked together by peptide bonds (also Peptides are molecules consisting of 2 to 50 amino acid residues a peptide bond, then the product is called called amide bonds). When two amino acid residues are linked through bonds to form a tripeptide; similarly, amino acids can a dipeptide. Three amino acids can be joined by two peptide be linked to form tetrapeptides, pentapeptides and so forth. Non-ribosomically synthesized peptides (i.e., Peptidess may be ribosomically and non-ribosomically synthesized. catalyzed by peptidyi transferase non-ribosomal peptides) are not specified by codons of mRNA and synthesis is not and their absolute configuration may of ribosomes. The nature of amino acids may be standard or non-standard decapeptides (peptides containing 10 amino be L- or D-. These peptides may be cyclic or linear in nature. Cyclic of non Bacillus brevis are examples acid residues) such as gramicidin S and tyrocidine produced by the bacterium residues. Ribosomically ribosomal peptides. Both peptides are antibiotics and contain both D- as well as L-amino acid is catalyzed by synthesized peptides (i.e., ribosomal peptides) are specified by codons of mRNA and their synthesis configuration. peptidyl transferase of ribosomes. The nature of amino acids is always standard and with L-absolute oxytocon and Small peptides (both nibosomal and non-ribosomal) play many roles in organisms. Some, such as others, vasopressin, are important hormones. Others, like glutathione, regulate oxidation-reduction reactions. Stil! such as enkephalins, are naturally occurring painkillers. Aspartame (L-aspartylphenylaianyB methyiester) is a commercially synthesized dipeptide and is used as an artificial sweetener. When many (generally >50) amino acid residues are joined, the product is called a polypeptide. Poiypeptides svnthesis occurs only ribosomicaly. Amino acids that have been incorporated into a peptide or poly peptide are termed amino acid residues. By convention, in a linear peptide or polypeptide, the left end is represented by the first amino acid while the right end is represented by the last anino acid. The first amino acid is also called N-term1na amino acid residue and the last amino acid is called the C-terminai amino acid residue. H H O H O N-terminal H,N-c-c- N -C -c N-C-c-o C-terminal R R Amino acid Amino acid Amno acd residue residue resdue Figure 1.11 Three amino acids joined by peptide bonds form a trpepbde, and each amino acd unt in troeptde s caied a residue. Alinear peptide or polypeptide chain has polarity bec3use ts ends are aferent, with an a-amno group at one end and an e-cartboxy! group at the other. The peptide bonds in proteins are formed between the aram1no and the a-carboxyi groups. But peptides oo occur naturaly where the peptide linkage involves a carboxy! or amino group which is attached to a carbon atom other than the a-carbon. For example, a dipeptide formed between the rcarboxyl group of glutamic acid an the amno group of alanine is called -giutamylalan1ne. Determination of molecular weight of peptides and polypeptides Molecular welght (or relative molecular mass, denoted M) of a substance is defined as the ratio of the mass of a molecule of that substance to one-tweifth the mass of carbon- 12 (C). Snce molecuiar werght s a ratio, it is dimensionless-it has no associated units. The average molecular weight of a standard amuN acd is nearer to 128. Although the average molecular weight of the 20 standard amino 3cds is about 138, the smaller amino ads predominate in most proteins. If we take into account the proportions in which the vanous amino aias occur in an average protein, the average molecular weght of protein amno acds is nearer to 128. Aiternattvey, this quantity may be expressed in tems of molecular mass. This is Simply the maSs of one moleule. or the molar mass divided by Avogadro's number. lt is expressed in units of daitons (Da) When an amino aOd partcipates in the formation of a polypeptide one moBecuie of water (M 18) s removed dunng peptide bond formation. Thus, the average mote ular weight of an aminoacid residue in a polypeptide is considered about 110(128-18).  Biomolecules and Catalysis 18 Problem The length of the protein X is 1000 amino acids and fluorescent protein (GFP). the molecular fused with green A protein X was mass of the fusion protein in approximate molecular daltons (Da)? GFP is 27 kDa. What is the total mass of s (in Da) = 1000 x 110 The approximate molecular mass of the protein x Solution: 110000 Da + 27000 of the fUsIon protein (n Da) = mass The total approximate molecular Problem then what Would be the molecular mass (in of 174 Daltons, Daltons) of a molecular mass a circular If one arginine has polymer of 38 arginines? molecular mass of amino acid qiven, so we will not take the average molecular mass is Solution: First, in this problem, circular polymer of 38 arginines 38 of acid residue. Second, during the formation of peptide bonds will form and formation amino water molecules. So. molecular mass of circular polymer of involve the release of 38 38 arginines of 38 peptide bonds - (38 x 18) = 6612 - 684 = 5928 Da. = (38 x 174) Poblem carboxyl-terminal are. Anentide has the sequence Glu-His-Trp-Arg-Gly. Tne pPAa Vaiues or tne dmino-terminal, respectively. Calculate the not those of the R aroups of the Glu, His and Arg are 9.6/, 2.34,.25, and 12,48, pH 3, 8 and 11?? Glu His Trp Arg GlyCOOH Net charge Solution NH, +1 -1 +2 +1 +1 pH 3 +1 -1 pH 8 +1 -1 +1 -1 -1 pH 11 -1 Problem at N-terminue A peptide has the sequence Glu-His-Trp--Ser-Arg-Pro-Gly. The pka Values of the a-amino group are 9.67, 2.34, 4.25. 6 and 12 0 a-carboxyl group at C-terminus and those of the R groups of the Glu, His and Arg respectively. Caiculate the pI of this peptide? Solution: To estimate the pi for this peptide, we will find net charge at pH 2.34, 4.25, 6.0, 9.67 and 12.48. The net charge at pH 1 will be +3. With increasing pH net charge on peptide will be 2.3 4.2 6.0 9.7 12.5 +3 +2 +1 -1 -2 Hence, pl = (9.67 +6)/2 = 7.8. 1.1.6 Peptide bond Ribosomal peptides and polypeptides are linear, unbranched polymers composed of amino acids linked together by peptide bonds. Peptide bonds are amide bonds formed between the a-amino group of one amino acid and the a-carboxyl group of another. This reaction is a dehydration reaction; that is, a water molecule is removed. The linked amino acids are referred to as amino acid residues. The peptide bond exhibits partial double bond character (approximately 40 percent) due to resonance. H H H,O HO=0 H H,NC-c -OH HNCC -OH H,N-CC- NC-C-OH J R H R, R H R the amino acid to Figure 1.12 The formation of a peptide bond (also called an amide bond) between the one a-carboxyl group of a-amino group of another amino acid is accompanied by the loss of a water molecule.  Biomolecules and Catalysis 19 carbon, pushing unhybridized lone pair of electrons to the carbonyt This arises because nitrogen can donate its electrons become the carbonyl double bond towards the oxygen, forming the oxygen anion. As a result, electrons from peptide bond. imparting partial double bond character to the delocalized over nitrogen, carbon, and oxygen atoms, the expected values characteristic contributes to the peptide bond length being only 1.33 , which lies between This the oxygen carries double bond (1.27 A). Due to electrons delocalization, for a C-N single bond (1.49 Å) and a C=N electric dipole. The partial a partial negative charge, while the nitrogen has a partial positive charge, creating a small linked by rigid, planar configuration. In a pair of amino acids double bond character maintains the peptide bond in a known as the amide plane. peptide bond, Six atoms (Cu, C, O, N, H and C,) lie in the same coplanar structure, a b C. a. :0: A m i dp el a n e H H H resonance. The peptide bond isa resonance hybrid Figure 1.13 Each peptide bond possesses partial double-bond character due to shown in (c). The dashed line indicates the resonance of of two states (a and b). The resonance structure of the peptide bond is peptide group lie in a single plane: the the peptide bond. For a pair of amino acids linked by a peptide bond, the six atoms of the a-carbon atom and co group of the first amino acid, and the NH group and a-carbon atom of the second amino acid. Due to the partial double-bond character, two possible configurations, cis and trans, are observed for a peptide bond in polypeptides. In the cis configuration, successive a-carbon atoms are on the same side of the peptide bond. In the trans configuration, the two successive a-cartbon atoms are on opposite sides of the peptide bond (figure 1.14). The restricted rotation about C-N bond can be specified by torsion angle (omega). In the trans configuration, o = 180° and in the cis configuration, o = 0°. H trans configuration (o = 180°) cis configuration (a = 0°) Figure 1.14 cis and trans configuration of peptide bond. In cis configuration, successive C, atoms are on the same side of the peptide bond whereas in trans configuration, successive C, atoms are on opposite sides of the peptide bond joining them. Virtually, all peptide bonds in proteins occur in trans configuration. The cis configuration is less stabie than the trans configuration. This preference for trans over cis can be explained by the fact that steric clashes between groups attached to the a-carbon atoms hinder the formation of the cis confiquration but do not arise in the trans configuration. However, this steric interference is reduced in peptide bonds to proline residues. In contrast with the peptide bond, the bonds between the nitrogen of amino group and the a-carbon atom (i.e., N-C, bond) and between the a-carbon atom and the carbon of carbonyl group (i.e., C-C bond) are pure singie bonds. The two adjacent rigid peptide units can rotate about these bonds, taking on various orientations. The rotations about these bonds can be specified by torsion angles (phi) and v (psi). The torsion angle about the bond between the amino nitrogen and the a-carbon atoms (N-C bond) is called bond between the a-carbon and the carbonyl carbon atoms tC-C whereas, the torsion angle about the bond) is caled. and v can have any value between +180° and -180° (i.e., 360 of rotation for each). But not all clashes of atoms in combinations are possible due to physical 3-dimensional space. Atoms take up space and two atoms cannot occupy the same the same time. These physical clashes are called space at steric interference. 20 Biomolecules and Catalysis Amp idle ane Know about convention To determine the and y angles between peptide planes, viewers should themselves at the Ca Carbon lookina imagine outward and should imagine startina from the = 0°, y = 0° conformation. From this perspective, positive values of correspond to cloCkwise rotations Go about the N-C, bond of the plane that includes the adjacent N-H group. Similarly, positive values of y correspond to clockwise rotations about the C-C bond of the plane that includes the adjacent C=O group. Both and y are defined as ±180° when the polypeptide =180° Amideplane is fully extended and all peptide groups V= 180 are in the samne plane. Figure 1.15 Two planar peptide groups are shown. The bonds between the amino nitrogen and the a-carbon atom (i e M bond) and between the a-carbon atom and the carbonyl carbon (i.., C-C bond) are pure single bonds. Hence, rotation can occur about these tbonds, The rotations about these bonds can be specified by torsion angles (phi) and y(psi). The torsion angle about the N-C, bond is called and that about the C-C bond is y. Most values of è and y are not allowed due to steric interference between atoms in the polypeptide backbone and aminoacid side chains. The combinations of ¢ and y values that are permitted in a peptide backbone or that are not permitted due to steric constraints were first determined by G. N. Ramachandran. These permitted values can be visualized on a two-dimensional plot called a Ramachandran plot. Polypeptide conformations are defined by the values of ¢ and y. Most values of ¢ and y are not allowed due to steric interference between non-bonded atoms. Hence, most areas of Ramachandran plot (i.e., most combinations of ¢ and y) represent sterically disallowed conformations of a polypeptide chain because of steric collisions between side chains and main chain. 180 180 90 90 (degrees) (degrees) V y -90 -90 A. B. -180 -180 -180 -90 90 180 90 180 -180 -90 (degrees) (degrees) Figure 1.16 A. Ramachandran plots showing allowed y are not combinations of the torsion angles and y. Most values of anndallowedÙand allowed due to steric interference between non-bonded atorms. The areas shaded dark black represent the sterically for branched y values. The plots for L-amino acid residues with unbranched side chains are nearly identical. The allowed ranges residue, which Is residues such as threonine are somewhat smaller than amino acids with chains. The glycine valuestoangles less sterically hindered, has a much broader allowed unbranched side range. The cyclic side chain of proline limits its range of except of around -60°, making it most types conformationally restricted amino acid in for all residue glycine. Each point represents and s values for an amino acid residue residue. B. Observed a well-refined x-ray values structure high resolution.  Biomolecules and Catalysis 21 disallowed conformations in which any non-bonding In the figure 1.16, the white regions correspond to sterically regions are sterically disallowed for interatomic distance is less than its corresponding van der Waals radii. These The black regions called allowed regions all amino acids except glycine which is unique as it lacks a side chain. and y values associated with each correspond to conformations where there are no steric interferences. Most of of the Ramachandran plot. There are, amino acid residue of a polypeptide chain fall within these allowed regions smallest side chain) is much less however, some notable exceptions. For example, glycine (the amino acid with range of and y covers a larger area sterically restricted than the other amino acid residues. Hence, its allowed its range of values to angles of around of the Ramachandran plot. In case of proline, the cyclic side chain limits regions correspond to conformation -60°, making it most conformationally restricted amino acid residue. Grey closer together. having outer limit van der Waals distances i.e., the atoms are allowed to come a little 1.1.7 Protein structure four levels of the Proteins are unbranched polymers constructed from 22 standard a-amino acids. They have As the structural organization. Primary structure, the amino acid sequence, is specified by genetic information. secondary polypeptide chain folds, it forms certain localized arrangements of adjacent amino acids that constitute structure. The overall three-dimensional shape that a polypeptide assumes is called the tertiary structure. Proteins that consist of two or more polypeptide chains (or subunits) are said to have a quaternary structure. Primary structure The primary structure (1° structure) of a polypeptide is its amino acid sequence. The arnino acids are connected by peptide bonds. Each polypeptide has its own unique sequence of amino acids. Primary structure of polypeptide determines to alarge extent the native (most frequently occurring)secondary and tertiary structures. Problem How many different pentapeptides from five amino acids (Gly, Asp, Tyr, Cys and Leu) are theoretically possible with no restrictions on the number of times a given residue type can be used. Solution: Suppose, 'X' is the number of amino acids used to prepare the peptides and 'n' is the number of amino acid residues in the peptide. If repetitions are allowed and order matters then we can calculate number of different penta peptides by using formula = x^. In this way, the number of different pentapeptides theoretically possibie ares i.e., 625. Probiem How many different pentapeptides are theoreticaltly possible that contain one residue each of Gly, Asp, Tyr, Cys and Leu? Solution: Suppose, "x* is the number of amino acids used to prepare the peptides and 'n' is the number of amino acid residues in the peptide. If no repetitions are alowed and order matters then we can calculate number of different pen tapeptides by using formula =X/(X - n)!. In this way, the number of different pentapeptides theoreticaly possible are 5!/ (5 - 5)! i.e., 120. Problem If there are 20 different standard amino acids and they can be assembled in any order, then how many different polypep tides of 100 amino acid residues can theoretically be produced? Solution: In general, the number of possibBe polypeptides for a sequence of n amino acids is the number of amino acids used to prepare the polypeptides raised to the power of n (i.e., 20^). Hence, a polypeptide with 100 amino acid residues, the number of different polypeptides that can be made from the 20 amino acids is 20!00 Secondary structure Protein secondary structure describes the spatial arrangement of its main-chain atoms, without regard to the positioningof its side chains. These structures are stabilized by hydrogen bonds between the carbonyl the amide hydrogen present in the polypeptide's backbone. oxygen and  Biomolecules and Catalysis 22 R R Polypeptide backbone (main chains) H H R R Secondary structures may have repetitive and regular patterns (torsion angle and y remains the same or nearly the unique structure (composed of sequences of residues throughout the segment) or iregular and which do not same values). The most common regular secondary structures are the a-helix and the B-sheet. The have similar o and y pattern is sometimessreferred to as coil (sometimes referred dto as secondary structure without a regular 'random coil"). + Helical conformations e.g., 2.2, helix, 3,, helix, Regular structure 3.6,, helix, 4.4,, helix Torsion angle and y remains + Pleated conformations the same or nearty the same Secondary structure throughout the segment of protein Irregular structure Do not have similar (Unique structure) and y value throughout the segment q-helix polypeptide chain twists into a helical conformation The The a-helix is a rigid, rod-like structure that forms when a (counterclockwise). Screw sense describes the screw sense of a-helix can be right-handed (ciockwise) or left-handed down the axis of a helix, the chain direction in which a helical structure rotates with respect to its axis. If, viewed is counterclockwise, the screw sense turns in a clockwise direction, it has a right-handed screw sense. If the turning is less steric clash between is left-handed. Right-handed helices are energetically more favorable because there of left the side chains and the backbone. Essentially all a-helices found in proteins are right-handed. Short regions per turn of handed a-helices (3-5 residues) occur only occasionally. In a-helix, there are 3.6 amino acid residues residue is related to the helix and the pitch (the length of one complete turn along the helix axis) is 0.54 nm. Each the next one by a rise of 1.5 Å (0.15 nm) along the helix axis. A single turn of a-helix involves 13 atoms from O to the Hof the hydrogen-bonded loop. For this reason, the a-helix is referred to as the 3.613-helix. -H-bonded loop H H13 n3 n+5 Ca ¿ C. n+4 H H-bonded loop Figure 1.17 The H-bond arrangement in the u-helix. AH-bond is formed between the Co of residue n and the NH of residue n+4. e a-helix is known as the 3.6,9-helix, where 3.6 is the numtber of residues per turn and 13 is the number of atoms in the H-bonded louy Length of u-helix is usualy 10-15 amino acid residues. In globular proteins, a-helices vary considerably in leg ranging from four or five to over forty amino acid residues. The average length is around ten residues, corresponding main to three turns. The a-helix is stabilized by intrachain of the hydrogen bonds between the NH and CO groups is situated chain. The CO group of each amino acid forms a hydrogen bond with the NH that group of the amino acidmain-chainCOand four residues ahead in the sequence. Except for amino acids near the u-helix, all the hydrogen ends of an NH groups are hydrogen bonded. In atypical u-helix of n AI the residues, there are n-4 hydrogen bonds. extendoutward bonds lie parallel to the helix axis and point in the same direction. The side chains of amino acids proline. from the helix. The amino acid side chains project out from exceptfor the u-helix and do not interfere with it,  Biomolecules and Catalysis 23 O H H 3.6 residues/turn 0.15 nm Pitch = 0.54 nm Axis Axis Figure 1.18 a. Describing the geomety of a-helix. The helix structure is defined by: the pitch (the distance along the axis between successive turns) and the rise per residue. The number of residues per helical turn is 3.6. In the right handed a-helx, a complete turn of the helix contains 3.6 amino acid residues, and the distance it rises per turn (its pitch) is 0.54 nm. b. The a-helix is stab1lized by intrachain hydrogen bonds between the NH and CO groups of the main chain. All the hydrogen bonds lie parallei to the hel1x axis and point in the same direction. The Rgroups of each amino acid residues in an a-helix face outward (not shown in the figure). Helices can be formed from either D- or L-amino acids, but a given helix must be composed entirely of amino acids of one configuration. The a-helix cannot be formed from a mixed copolymer of D- and L-amino acids. Amino acids also have different propensities for forming a-helices. Amino acids such as alanine, glutam1ne, glutamate, leucine, methionine, arginine show the higher tendency to form a-helices (helx former). Branching at the -carbon atom, as in valine, threonine and isoleucine, tends to destabilize a-helices because of steric clashes (helix breaker). Serine, aspartate and asparagine also tend to disrupt a-helices because their side chains contain hydrogen-bond donors or acceptors in close proximity to the main chain, where they compete for main-chain NH and CO groups. Proline also is a helix breaker and least commonly found in a-helices. It is an imino acid; it has a secondary nitrogen with only one hydrogern. When participating in a peptide bond, the nitrogen no longer has hydrogen bound to it. Thus, peptide bonds in which the nitrogen is supplied by proline are incapable of functioning as Secondly, its ring structure prevents it from assuming the value to fit into an a-helix. hydrogen-bond donors. Apart from most common a-helix (3.613 helix), other helical structures (such as 2.2, are also found in proteins. The second most helix, 310 helix and 4.416 helix) common helical structure is the 3,0 helix, which has three residues per turn and 10 atoms in the hydrogen-bonded loop. Type 2.2, helix 310 helix 3.61 helix 4.416 or helix Residues per turn 2.2 3.6 4.4 Atoms in H-bonded loop 7 10 13 16 Hydrogen bonding n-n +2 n-n+3nn +4 n+ 5 B-pleated sheets B-pleated sheets (or, more simply, the B-sheet) form when two or more polypeptide side. Each individual segment is referred to as a B-strand. Rather than being coiled, chain segments line up side by The distance between adjacent amino each B-strand is fully extended. acids along a -strand is of 1.5 A along an u-helix. B-pleated sheets are approximately 3.5 A, in contrast with a distance polypeptide backbone N-H and carbonyl groups stabilized of by interchain hydrogen bonds that form between the adjacent strands.  Biomolecules and Catalysis 24 B-sheet Figure 1.19 B-sheets are formed from extended ß-strands. Hydrogen bonding occurs between neighborina B-strands rather than within one as in an a-helix. ß-sheets in proteins contain 2 to as many as 22 B-strand ß-strands. with an average of 6 p-strands. Each strand may contain up to 15 residues, the average being 6 residues. neighboring B-s-strands rather than within one however, hydrogen bonding occurs between as in an al In B-sheets, sheet structuree or antiparallel. In parallel B-pleated g-helix. Adiacent strand can be either parallel B-pleated | sheet structures, theB-strands run in opposite arranged in the same direction. However, in antiparallel bonde fo are parallel -Sheets because rully collinear hydrogen directions. Antiparallel B-sheets are more stable than and some antinarallal B-sheets with some B-strands pair parallel B-strands can also combirne into mixed Antiparallel B-strands H n+2 n+4 Cn+1 N-terminus n+3 C-terminus Ca N Co C-terminus N n+3 n+1 N-terminus n+4 n+2 H Parallel Bstrands n+2 N-terminus n+1 C-terminus C H N-terminus C-terminus C N C n+2 N n+3 n+1 H distinctive pattern the two forms has a betweenNH and each of Figure 1.20 p-stranas can interact in ether paraliel or ant1parallel orientat1on bonds directions. Hydrogen sheet, adjacent of hydrogen bonding. In an entiparalle -pieated sheet, adjacent strands rurn in opposite parallel B-pleated acidsonthe and CO groups connect each amio aCAd to a sangie afmsno aid on an adjacent strand. In the amino adjacentstrand two different strands run in the same direcion. tydroger bonds connect each arnino acid on one strand with the amino acid on adjacent strand. For each amino acid, the Nh groups hydrogen bonded to the CO group of one the chain. whereas the CO group is hydrogen bonded to the NH group on the amino acidtwo residues farther along Problem You have a polypeptide chain containing 105 arino acid residues. 1. calcuiate the approximate length (in nanorneters) if it exists entirely in u-heical form. 2. calculate the approximate length (in nanometers) if it exists entirely in futly extended forn. a-helicatfor 3. calculate the number of H-bonds invoBving the tbackbone CO and NH f it ex1sts in entirely  Biomoler ules and Catalysis 25 adjacent amino acid extended conformation. The distance between Solution: As we know, B-strand represents a fully contrast with a distance of 1.5 Åalong an a-helix. residues along a B-strand is approximately 3.5 Å, in 1. 105 x 1.5 Å= 157.5Å= 15.75 nanometers. 2. 105 x 3.5 Å = 367.5 Å = 36.75 nanometers. make a maximum of two H-bonds. Therefore 105 residues can make up to 3. In an a-helix, one amino acid residue can C-terminus make 2 x 105 = 210 H-bonds. In the a-helix, four amino acid residues at the N-terminus and four at the number of H-bonds 210 - 8 = 202. When calculating this number, only 1 H-bond per residue. This makes the total number of H-bonds and one for the acceptor. Hence, the actual we count each H-bond twice: one for the donor residue is 202/2 = 101. characteristics and y values. Each type of Common regular secondary structures such as a-helix, B-sheet have associated with each amino acid residue. The regular secondary structure can be described by the and y values respect to the Co group, they have D- and L-form of the amino acids have their side-chain oriented differentiy with y angles than different allowed and y angles. If polypeptides are buitt from D-amino acids, they have different and (figure 1.21), the torsion those that are exclusively made up of L-amino acids. As shown by a Ramachandran piot angles that define the a-helix and B-sheet fall within a relatively restricted range of stericaily aliowed regions. The backbone torsion angles for right-handed a-helix fall in the plot's lower left quadrant. In contrast, B-sheet is made up of almost fully extended strands, with and y angles fall in the upper left quadrant of the Ramachandran plot. 180 180 90 90 (degrees) (degrees) v v -90 -90 -180 -180 -180 -90 90 180 -180 -90 180 (degrees) o(degrees) Figure 1.21 Ramachandran plots showing a variety of secondary structures. The vaiues of and v for various iowed secondary structures are overlaid on the plot. The white circles represent torsion angles of several secondary helix; u, for teft-handed a-helix; 3for right-handed 3,-helix; 2 for 2.2,-helix: C for structures tor rnght-handed coltagen, for paralet $-sheet and. tor antiparaltet -sheet. Table 1.4 ldealized and y angles for common secondary structures in protens Torsion angle (in degree Secondary structure Torsion angle (i degree) Secondary structure Antiparalei -sheet -139 + L35 Right-handed 3,o-hetix 49 26 Parallel sheet -19 Coitagen + 153 Right-handed 3.6,, heltx 52 47 Lert-handed a -hex 47 Note: In reai poteins, Uhe torsion anghes often vary somehat frorn chese dealizet vaiues  Bomoleules andCatalsis Turns The - -helix and the Secondary struclures are uSually divided into n-helix, t-sheet and loops. seCondry structures. These secondary structures have repetitive and regular patterns -sheet are reqular (dihedral remains the same or nearly the same throughout the segment). As compared to a-helix and (-sheet, , angle and w have regular, penodic structures. They have non-repetitive, nonregular secondary of residues which do not have similar andy values. Loops interconnect different structure composed loops do not of secondary structures andsequences change the drection of the polypeptide cthain. They are typically found on the surface of the globular largely respons1ble for its shape and dynamics. protein, which is Shorter loops are usually called turns. Turns are composed of three to six amino acid residues. The turns can be yturns(cons1sting of 3am1no acid residues), p-turns(four amino acid residues), u-turns (five and t-turns (siN amino acid residues). Ahydrogen bond betweentheir end residues stabilizes these acid amino residues) secondary structures. Glyine and proline are commonly present in turns. The lack of alarge side short, chain in a U-shaped and stabiization of the cis configuration of the peptide bond by proline allow the polypeptide backbone to fola a tight U-shape. Turns altow large proteins to fold into highly compact structures. Turns are classified usuaily in terms of andv angles or the two end residues participating in hydrogen bondies In an a-tun, the hydrogen bond donor and acceptor residues are separated by four peptide bonds (which invalvee five amino acid residues). H-bond forms between the carbonyl oxygen of residue (n) and the hydrogen of the amide group of residue (n+4). -tum (or reverse turn or hairpin turn), the most common form, is characterized by hydrogen bond(s) in which the donor and acceptor residues are separated by three peptide involves four amino acid residues, with the carbonyl oxygen of the first residue bonds. The structure forming a hydrogen bond with the amino-group hydrogen of the fourth (n and n+3). The peptide groups of the central two residues do not in any hydrogen bonding. There are several types of participate B-turns, each defined by the and y angles of residues n+1 and n+2. Type iand II B-tuns are the most common, and they differ mainly in the orientation of the peptide bond between the residues n+1 and n+2. n+2 Tt+1 H n+3 Tyoe i -turn Type Il B-turn Figure 1.2 Struture cf a turn The CO group of resCue n of the residue R5 to stabiize the turm -turns nave been dassified poBypeptde chain is hydrogen bonded to the NH group o and v, Le n l and -2 according to the values of their central residue dihedral angles. Similariy, à y-turn rs charactenzed by hydrogen bond(s) in which the donor and To peptioe bonds. Itis a three amino acd res.due turn acceptor residues are separated oy with a hydrogen bond between the first and third resioues ( and n+2). It is a three aminc acid resadue turn with a hydrogen bond between the first and third residues -turn $-turn y-turn Frve amino acid residues Four am1no acd residues Three amino acId residues Four peptide Dords Three peptide bonds Two peptide bonds H-bond between n and n+4 H-bond between n and n+3 H-bond betweenn and n+2  BIomolecules and Catalysis 27 Box 1.2 Motif and domai