1. Basic protein structure and stability I: Formation of peptide bonds/ properties of amino acids Biochem 565, Fall 2008 08/25/08

2. 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

3.  -amino acids-- the building blocks of proteins

4. The protein alphabet--the 20 amino acid R groups

5. Aromatic ring numbering/naming (IUPAC) IUPAC nomenclature: http://www.chem.qmw.ac.uk/iupac/AminoAcid/index.html

6. Proteins are made by controlled polymerization of amino acids

7. 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

8. 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 70-100 residues in length (chemical ligation can be used to make longer ones)

9. 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

10. 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

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

12. Sizes of amino acids a.avol (Å 3 )surface area(Å 2 ) A88.6115 R173.4 225 D111.1150 N114.1160 C108.5135 E138.4190 Q143.8180 G 60.1 75 H153.2195 I166.7175 L166.7170 K168.6200 a.avol (Å 3 )surface area(Å 2 ) M162.9 185 F189.9210 P112.7145 S 89.0115 T116.1140 W227.8255 Y193.6230 V140.0155 volume: Zamyatin A Prog Biophys Mol Biol 24, 107 (1972) surface area: Chothia C J Mol Biol 105, 1 (1975)

13. Acid-base titration curves of ionizable side chains 3 4 5 6 7 8 9 10 11 12 13 14 Arg+ Lys+ Tyr Cys His+ Asp and Glu pH eq. OH - added 1 0 pK a physiological pH acid base

14. The basic side chains pct occurrence in proteins

15. The acidic side chains

16. Shifting of side chain titration curves 3 4 5 6 7 8 9 10 11 12 13 14 His+ pH eq. OH - added 1 0 pK a physiological pH acid base

17. 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

18. 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, 105-32 (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

19. The aliphatic amino acids (plus methionine)

20. Aromatic side chains

21. The polar uncharged side chains

22. 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

23. 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

24. 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

25. Grouping the amino acids by properties from http://www.russell.embl-heidelberg.de/aas/ which adapted it from Livingstone & Barton, CABIOS, 9, 745-756, 1993.