Basic protein structure and stability I: Formation of peptide bonds/ properties of amino acids Biochem 565, Fall /25/08

MQTLSERLKKRRIALKMTQTELATKAGVKQQSIQLIEAGVT KRPRFLFEIAMALNCDPVWLQYGTKRGKAA atgcaaactctttctgaacgcctcaagaagaggcgaattgcgttaaaaatgacgcaaaccgaa ctggcaaccaaagccggtgttaaacagcaatcaattcaactgattgaagctggagtaaccaa gcgaccgcgcttcttgtttgagattgctatggcgcttaactgtgatccggtttggttacagtacgg aactaaacgcggtaaagccgcttaa augcaaacucuuucugaacgccucaagaagaggcgaauugcguuaaaaaugacgcaaacc gaacuggcaaccaaagccgguguuaaacagcaaucaauucaacugauugaagcuggagua accaagcgaccgcgcuucuuguuugagauugcuauggcgcuuaacugugauccgguuug guuacaguacggaacuaaacgcgguaaagccgcuuaa Proteins are the primary functional manifestation of the information in genomes DNA sequence RNA sequence protein sequence protein structure protein function transcription translation

 -amino acids-- the building blocks of proteins

The protein alphabet--the 20 amino acid R groups

Aromatic ring numbering/naming (IUPAC) IUPAC nomenclature:

Proteins are made by controlled polymerization of amino acids

Solid phase peptide synthesis (SPPS) solid support fmoc protecting group protecting groups for side chains 1st and 2nd amino acids carbonyl activating group adapted from Sigma-Aldrich website

Solid phase peptide synthesis (SPPS) at the end a final deblocking is done followed by removal of the side-chain protecting groups and cleavage from the resin to recover the peptide SPPS using Fmoc can be used to make peptides up to residues in length (chemical ligation can be used to make longer ones)

Peptide bond formation in vivo aminoacyl t-RNA ester activates carbonyl, making peptide bond formation favorable adenine 2451 of 23S ribosomal RNA abstracts proton from amino group, catalyzing nucleophilic attack chemical protecting groups are not necessary because the ribosomal machinery ensures selective positioning and activation of the reactants t-RNA

Peptide bond formation in vivo peptidyl t-RNA shifts to P-site new aminoacyl t-RNA comes into A-site deacylated t-RNA leaves P-site

Properties of the amino acid side chains size acid-base equilibria hydrophobicity/polarity tautomerism oxidation/reduction of cysteine chemical reactivity (next lecture)

Sizes of amino acids a.avol (Å 3 )surface area(Å 2 ) A R D N C E Q G H I L K a.avol (Å 3 )surface area(Å 2 ) M F P S T W Y V volume: Zamyatin A Prog Biophys Mol Biol 24, 107 (1972) surface area: Chothia C J Mol Biol 105, 1 (1975)

Acid-base titration curves of ionizable side chains Arg+ Lys+ Tyr Cys His+ Asp and Glu pH eq. OH - added 1 0 pK a physiological pH acid base

The basic side chains pct occurrence in proteins

The acidic side chains

Shifting of side chain titration curves His+ pH eq. OH - added 1 0 pK a physiological pH acid base

Poorly populated but highly reactive forms of amino acids base form of lysine not highly populated in general at physiological pH, but is a reactive nucleophile, and if present even in minuscule amounts may do chemistry

Kyte-Doolittle hydropathy of amino-acid residues side chainhydropathy index Ile4.5 Val4.2 Leu3.8 Phe2.8 Cys2.5 Met1.9 Ala1.8 Gly-0.4 Thr-0.7 Trp-0.9 side chainhydropathy index Ser-0.8 Tyr-1.3 Pro-1.6 His-3.2 Glu-3.5 Gln-3.5 Asp-3.5 Asn-3.5 Lys-3.9 Arg-4.5 Kyte J & Doolittle RF J Mol Biol 157, (1982) Many attempts have been made to quantify polarity, nonpolarity (hydrophobicity) of amino-acid residues in terms of scales. Kyte-Doolittle is a classic one. It is based on transfer free energies from nonpolar solvents to water combined with measurements of the tendency of residues to be buried in proteins. nonpolar--blue; polar--red; ambiguous--purple

The aliphatic amino acids (plus methionine)

Aromatic side chains

The polar uncharged side chains

Histidine--the “ambidextrous” side chain acid base pK a ~ 7  Histidine is just barely acidic enough to populate base forms at neutral pH  therefore, its base form is about the strongest base that can exist under physiological conditions  the base form has two tautomers: one nitrogen can act as a base/ nucleophile, while the other can act as a hydrogen donor-- ”ambidextrous” predominant form in model peptides

Cysteine and cystine disulfide formation disulfide exchange disulfide exchange occurs through the thiolate anion at neutral to basic pH Pairs of cysteines frequently undergo oxidation to a disulfide bonded form called “cystine” more hydrophobic than cysteine

amino acids don’t fall neatly into classes--they are different combinations of small/large, charged/uncharged, polar/nonpolar properties how we casually speak of them can affect the way we think about their behavior. For example, if you think of Cys as a polar residue, you might be surprised to find it in the hydrophobic core of a protein unpaired to any other polar group. But this does happen. the properties of a residue type can also vary with conditions/environment Key points about the character of amino acid side chains

Grouping the amino acids by properties from which adapted it from Livingstone & Barton, CABIOS, 9, , 1993.