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

Levels of Protein Structure The AA sequence of a protein's polypeptide chain is called its primary structure. Different regions of sequence form local regular secondary structures, (a-helices or -strands). Tertiary structure is formed by packing 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 structures, amino acids far apart in the sequence can be brought closer together to form a functional region, called an active site.

Amino Acids Organic compounds with amino and carboxylate functional groups Each AA has unique side chain (R) attached to alpha (α) carbon Crystalline solids with high MP’s Highly-soluble in water Exist as dipolar, charged zwitterions (ionic form) Exist as either L- or D- enantiomers Almost without exception, biological organisms use only the L enantiomer Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7th Edition, 2011; Berg JM, Tymoczko JL, Stryer L, Biochemistry, 5th Edition, 2002

Formation of Polypeptides Polypeptides and proteins are created through formation of peptide bonds between amino acids Condensation reaction Peptide linkages Polypeptide http://en.wikipedia.org/wiki/File:AminoacidCondensation.svg

Polypeptide Chain 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.

The “Handedness" of Amino Acids. 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.

Amino Acids 20 amino acids specified by the genetic code, grouped by different properties associated with R group (residues) http://courses.bio.indiana.edu/L104-Bonner/Sp11/imagesSp11/L12/Part1MPs.html

Hydrophobic Amino Acids

Charged Amino Acids

Polar Amino Acids

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

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

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

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

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

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

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

Disulfide Bonds 2 -CH2SH + 1/2 O2  -CH2-S-S-CH2 + H2O Disulfide bonds form between side chains of two cysteine residues. Two SH groups from cysteine residues, which may be in different parts of the AA sequence but adjacent in the 3D structure, can be oxidized to form one S-S (disulfide) group. Usually occurs in extracellular proteins. 2 -CH2SH + 1/2 O2  -CH2-S-S-CH2 + H2O

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

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

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

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

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

The Catalytic Triad of Trypsin

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

Chemical Structure of Asp 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

Calcium-binding Site in Calmodulin

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

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

Chemical Structure of Gln 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 Lys 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 Arg 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 Guanidine group

Chemical Structure of His Histidine His H 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

Distribution of Amino Acids Codons (3 RNA bases in sequence) determine each amino acid that will build the protein expressed

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.

Resonance Tautomers of a Peptide

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.

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.

Torsion Angles Phi and Psi

Ramachandran Plots 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, and L correspond approximately to conformational angles found for the usual right-handed a helices, b strands, and left-handed a helices,respectively. Ramachandran Plots

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.)

Certain Side-chain Conformations are Energetically Favorable Think of the Newman projections that you learned in organic chem. 9780738041353 3 conformations of Val The staggered conformations are the most energetically favored conformations of two tetrahedrally coordinated carbon atoms.

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

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

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.

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.

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.