Chemistry 501 Handout 3 Amino Acids, Peptides, and Proteins Chapter 3

1. Dep. of Chemistry & Biochemistry Prof. Indig Chemistry 501 Handout 3Amino Acids, Peptides, and ProteinsChapter 3 Lehninger. Principles of Biochemistry. by Nelson and Cox, 5th Edition; W.H. Freeman and Company

2. Amino Acids Steric relationship of the stereoisomers of Alanine to the absolute configuration of L- and D-glyceraldehyde The amino acid residues in proteins are the L isomers

3. Amino acids can be classified by R groups

5. Reversible formation of disulfide bond by the oxidation of two molecules of cysteine e.g. two polypeptide chains of insuline

6. indole ring

7. Absorption of ultraviolet light by aromatic amino acids Lambert-Beer Law log (Io/I) = e C L

8. imidazole guanidino

10. prothrombim, a # of Ca+ binding proteins g b a 4 3 plant cell wall, collagen 1 5 2 elastin collagen Lysine residues myosin Uncommon amino acids also have important functions Residues created by modification of common residues already incorporated into a polypeptide ~ 300 additional amino acids have been found in cells rare, introduced during protein synthesis rather than created through a postsynthetic modification

11. Reversible amino acid modifications involved in regulation of protein activity

12. Amino acids can act as acids and bases Nonionic and zwitterionic forms of amino acids Titration of glycine Titration curves predict the electric charge of amino acids Isoelectric point (or isoelectric pH) pI= ½ (pk1 + pk2) = ½ (2.34 + 9.60) = 5.97 amphoteric (ampholytes - amphoteric electrolytes)

13. Effect of chemical environment on pKa

14. Amino acids differ in their acid-base properties pka of the –COOH group: 1.8 – 2.4 pka of the –NH3+ group: 8.8 – 11.0 Amino acids with R groups that do not ionize Amino acids with ionizable R groups e.g. three stages (three ionization steps  three pka values)

17. Peptides are chains of amino acids Pentapeptide condensation hydrolysis Serylglyciltyrosylalanylleucine or Ser-Gly-Tyr-Ala-Leu or SGYAL Two amino acid molecules can be covalently joined through a substituted amide linkage, termed a peptide bond, to yield a dipeptide Peptides are named beginning with the amino-terminal residue, which by convention is placed at the left. just a few residues  oligopeptide many residues  polypeptide

18. Biologically active peptides and polypeptides occur in a vast range of sizes If at least two chains are identical → the protein is said to be oligomeric, and the identical units (consisting of one or more chains) are referred to as protomers. Multisubunit proteins: have two or more polypeptide chains associated noncovalently The vast majority of naturally occurring proteins contain fewer than 2,000 amino acid residues.

19. Polypeptides have characteristic amino acid compositions Some proteins contain chemical groups other than amino acids (conjugated proteins). The non-amino acid part of the conjugated protein is usually called its prosthetic group.

20. Protein Separation and Purification Column Chromatography Crude extract --> --> --> fractionation

21. Ion-Exchange Chromatography Example: Cation-exchange chromatography

22. Exclusion Chromatography (gel filtration)

23. Affinity Chromatography

24. Refers to the total number of units of enzyme in a solution. A measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure. Number of enzyme units per milligram of total protein. 1.0 unit of enzyme activity = amount of enzyme causing the transformation of 1.0 mmol of substrate per minute at 25oC under optimal conditions of measurement.

25. Electrophoresis gel stained with a protein-specific dye (e.g. coomasie blue) SDS-polyacrylamide gel Purification of RNA polymerize from E. coli Cross-linked polymer polyacrylamide SDS CH3(CH2)11SO4-Na+ acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio.

26. Isoelectric focusing Two-dimensional electrophoresis

27. There are several levels of protein structure Multisubunit proteins Arrangement is space of polypeptide subunits Particularly stable arrangements of amino acid residues giving rise to recurring structural paterns All aspects of the 3-D folding of a polypeptide Includes disulfide bonds

28. Determination of amino acid sequence Amino acid sequence of bovine insulin (10 years of work by Sanger) The amino acid sequences of millions of proteins have been determined Identical in human, pig, rabbit and sperm whale Identical in cow, dog, goat and horse

29. Short polypeptides are sequenced using automated procedures Sanger’s method for identifying the amino-terminal residue The Edman degradation procedure (carried out on a sequenator) reveals the entire sequence of a peptide

30. Large proteins must be sequenced in smaller fragments Breaking disulfide bonds Cleaving the polypeptide chain Some proteases cleave only the peptide bond adjacent to particular amino acid residues

31. Ordering the peptide fragments Met (C)

32. Amino acid sequences can also be deduced by other methods codon Correspondence of DNA and amino acid sequences

33. Investigating proteins with mass spectrometry

34. Small peptides and proteins can be chemically synthesized

35. (9-fuorenylmethoxycarbonyl)