Chapter 1/Structure I

1. Chapter 1/Structure I The Building Blocks Chemical Properties of Polypeptide Chains

2. Level of Protein Structure The amino acid sequence of a protein's polypeptide chain is called its primary structure. Different regions of the sequence form local regular secondary structures, such as alpha (a) helices or beta () strands. The tertiary structure is formed by packing such structural elements into one or several compact globular units called domains. The final protein may contain several polypeptide chains arranged in a quaternary structure. By formation of such tertiary and quaternary structures, amino acids far apart in the sequence are brought close together in three dimensions to form a functional region, called an active site.

3. Amino Acids • Proteins are built up by amino acids that are linked by peptide bonds to form a polypeptide chain. • An amino acid has several structural components: • A central carbon atom (Ca) is attached to • an amino group (NH2), • a carboxyl group (COOH), • a hydrogen atom (H), • a side chain (R).

4. In a polypeptide chain the carboxyl group of the amino acid n has formed a peptide bond, C-N, to the amino group of the amino acid n + 1. One water molecule is eliminated in this process. The repeating units, which are called residues, are divided into main-chain atoms and side chains. The main-chain part, which is identical in all residues, contains a central Ca atom attached to an NH group, a C'=O group, and an H atom. The side chain R, which is different for different residues, is bound to the Ca atom. Polypeptide Chain

5. Looking down the H-Ca bond from the hydrogen atom, the L-form has CO, R, and N substituents from Ca going in a clockwise direction. For the L-form the groups read CORN in the clockwise direction. All a.a. except Gly (R = H) have a chiral center All a.a. incorporated into proteins by organisms are in the L-form. The “Handedness" of Amino Acids.

6. Hydrophobic Amino Acids

7. Charged Amino Acids

8. Polar Amino Acids

9. Glycine Gly G Glycine Relative abundance 7.5 % flexible, seen in turns Chemical Structure of Gly

10. Alanine Ala A Alanine Relative abundance 9.0 % hydrophobic, unreactive, a-helix former Chemical Structure of Ala

11. Valine Val V Valine Relative abundance 6.9 % hydrophobic, unreactive, stiff, b-substitution b-sheet former Chemical Structure of Val

12. Leucine Leu L Leucine Relative abundance 7.5 % hydrophobic, unreactive, a-helix, b-sheet former Chemical Structure of Leu

13. Isoleucine Ile I Isoleucine Relative abundance 4.6% hydrophobic, unreactive, stiff, b-substitution b-sheet former Chemical Structure of Ile

14. Methionene Met M Methionine Relative abundance 1.7 % thio-ether, un-branched nonpolar, ligand for Cu2+ binding a-helix former Chemical Structure of Met

15. Cysteine Cys C Cysteine pKa = 8.33 Relative abundance 2.8 % thiol, disulfide cross-links, nucleophile in proteases ligand for Zn2+ binding b-sheet, b-turn former Chemical Structure of Cys

16. Disulfide Bonds • Disulfide bonds form between the side chains of two cysteine residues. • Two SH groups from cysteine residues, which may be in different parts of the amino acid sequence but adjacent in the three-dimensional structure, are oxidized to form one S-S (disulfide) group. 2 -CH2SH + 1/2 O2 -CH2-S-S-CH2 + H2O

17. Proline Pro P Proline Relative abundance 4.6 % 2° amine, stiff, 20 % cis, slow isomerization seen in turns Initiation of a-helix Chemical Structure of Pro

18. Phenylalanine Phe F Fenylalanine Relative abundance 3.5 % hydrophobic, unreactive, polarizable absorbance at 257 nm Chemical Structure of Phe

19. Tryptophan Trp W tWo rings Relative abundance 1.1 % largest hydrophobic, absorbance at 280 nm fluorescent ~340 nm, exhibits charge transfer Chemical Structure of Trp

20. Tyrosine Tyr Y tYrosine pKa = 10.13 Relative abundance 3.5 % aromatic, absorbance at 280 nm fluorescent at 303 nm can be phosphorylated hydroxyl can be nitrated, iodinated, & acetylated Chemical Structure of Tyr

21. Serine Ser S Serine Relative abundance 7.1 % hydroxyl, polar, H-bonding ability nucleophile in serine proteases phosphorylation and glycosylation Chemical Structure of Ser

22. The Catalytic Triad of Trypsin

23. Threonine Thr T Threonine Relative abundance 6.0 % hydroxyl, polar, H-bonding ability, stiff, b-substitution phosphorylation and glycosylation Chemical Structure of Thr

24. Aspartic Acid Asp D AsparDic pKa = 3.90 Relative abundance 5.5 % carboxylic acid, in active sites for cleavage of C-O bonds, member of catalytic triad in serine proteases acts in general acid/base catalysis, ligand for Ca2+ binding Chemical Structure of Asp

25. Calcium-binding Site in Calmodulin

26. Glutamic Acid Glu E GluEtamic pKa = 4.07 Relative abundance 6.2 % carboxylic acid, ligand for Ca2+ bindingas acts as a general acid/base in catalysis for lysozyme, proteinase Chemical Structure of Glu

27. Asparagine Asn N AsparagiNe Relative abundance 4.4 % Polar, acts as both H-bond donor and acceptor molecular recognition site can be hydrolyzed to Asp Chemical Structure of Asn

28. Glutamine Gln Q Qutamine Relative abundance 3.9% Polar, acts as both H-bond donor and acceptor molecular recognition site can be hydrolyzed to Asp N-terminal Gln can be cyclized Chemical Structure of Gln

29. Lysine Lys K Before L pKa = 10.79 Relative abundance 7.0 % amine base, floppy, charge interacts with phosphate DNA/RNA forms schiff base with aldehydes (-N-N=CH-) a catalytic residue in some enzymes Chemical Structure of Lys

30. Arginine Arg R aRginine pKa = 12.48 Relative abundance 4.7 % Guanidine group, good charge coupled with acid charge interacts with phosphate DNA/RNA a catalytic residue in some enzymes Chemical Structure of Arg

31. Histidine His H Histidine pKa = 6.04 Relative abundance 2.1 % imidazole acid or base; pKa = pH (physiological), member of catalytic triad in serine proteases ligand for Zn2+ and Fe3+ binding Chemical Structure of His

32. Properties of the Peptide Bond • Each peptide unit contains the C atom and the C'=O group of the residue n as well as the NH group and the C atom of the residue n + 1. • Each such unit is a planar, rigid group with known bond distances and bond angles. R1, R2, and R3 are the side chains attached to the Ca atoms that link the peptide units in the polypeptide chain. • The peptide group is planar because the additional electron pair of the C=O bond is delocalized over the peptide group such that rotation around the C-N bond is prevented by an energy barrier.

33. Resonance Tautomers of a Peptide

34. Peptide Bond • The peptide bonds are planer in proteins and almost always trans. • Trans isomers of the peptide bond are 4 kcal/mol more stable than cis isomers => • 0.1 % cis.

35. Polypeptide Chain • Each peptide unit has two degrees of freedom; it can rotate around two bonds, its Ca-C' bond and its N-Ca bond. • The angle of rotation around the N-Ca bond is called phi (f) and that around the Ca-C' bond is called psi (y). • The conformation of the main-chain atoms is determined by the values of these two angles for each amino acid.

36. Torsion Angles Phi and Psi

37. Ramachandran plots indicate allowed • combinations of the conformational • angles phi and psi. • Since phi (f) and psi (y) refer to • rotations of two rigid peptide • units around the same Ca atom, most • combinations produce steric • collisions either between atoms in • different peptide groups or • between a peptide unit and the side • chain attached to Ca. These • combinations are therefore not allowed. • Colored areas show sterically allowed • regions. The areas labeled a, b, andL • correspond approximately to • conformational angles found for the • usual right-handed a helices, b strands, • and left-handed a helices,respectively. Ramachandran Plots

38. Calculated Ramachandran Plots for Amino Acids Gly with only one H atom as a sidechain, can adopt a much wider range of conformations than the other residues. • (Left) Observed values for all residue types except glycine. Each point represents f and y values for an amino acid residue in a well-refined x-ray structure to high resolution. • (Right) Observed values for glycine. Notice that the values include combinations of  and y that are not allowed for other amino acids. (From J. Richardson, Adv. Prot. Chem. 34: 174-175,1981.)

39. Certain Side-chain Conformations are Energetically Favorable • The staggered conformations are the most energetically favored conformations of two tetrahedrally coordinated carbon atoms. 3 conformations of Val

40. Side Chain Conformation • The side chain atoms of amino acids are named using the Greek alphabet according to this scheme.

41. Side Chain Torsion Angles • The side chain torsion angles are named chi1, chi2, chi3, etc., as shown below for lysine.

42. Chi1(χ1) Angles • The chi1 angle is subject to certain restrictions, which arise from steric hindrance between the gamma side chain atom(s) and the main chain. • The different conformations of the side chain as a function of chi1 are referred to as gauche(+), trans and gauche(-). These are indicated in the diagrams here, in which the amino acid is viewed along the Cb-Ca bond. The most abundant conformation is gauche(+), in which the gamma side chain atom is opposite to the residue's main chain carbonyl group when viewed along the Cb-Ca bond.

43. Gauche The second most abundant conformation is trans, in which the side chain gamma atom is opposite the main chain nitrogen. The least abundant conformation is gauche(-), which occurs when the side chain is opposite the hydrogen substituent on the Ca atom. This conformation is unstable because the gamma atom is in close contact with the main chain CO and NH groups. The gauche(-) conformation is occasionally adopted by Ser or Thr residues in a helices.

44. Chi2 (2) • In general, side chains tend to adopt the same three torsion angles (+/- 60 and 180 degrees) about chi2 since these correspond to staggered conformations. • However, for residues with an sp2 hybridized gamma atom such as Phe, Tyr, etc., chi2 rarely equals 180 degrees because this would involve an eclipsed conformation. For these side chains the chi2 angle is usually close to +/- 90 degrees as this minimizes close contacts. • For residues such as Asp and Asn the chi2 angles are strongly influenced by the hydrogen bonding capacity of the side chain and its environment. Consequently, these residues adopt a wide range of chi2 angles.

45. Many Proteins Contain Intrinsic Metal Atoms • (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules. • (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains.

46. EF-hand Calcium-binding Motif • The calcium atom is bound to one of the motifs in the muscle protein troponin-C through six oxygen atoms: one each from the side chains of Asp (D) 9, Asn (N) 11, and Asp (D) 13; one from the main chain of residue 15; and two from the side chain of Glu (E) 20. In addition, a water molecule (W) is bound to the calcium atom.