1. Chemistry 501 Handout 3 Amino Acids, Peptides, and Proteins Chapter 3 Dep. of Chemistry & Biochemistry Prof. Indig Lehninger. Principles of Biochemistry. by Nelson and Cox, 5 th 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 (I o /I) =  C L

8. guanidino imidazole

10. Uncommon amino acids also have important functions Residues created by modification of common residues already incorporated into a polypeptide 1 2 34 5  plant cell wall, collagen myosin prothrombim, a # of Ca + binding proteins elastin Lysine residues ~ 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. Titration of glycine Amino acids can act as acids and bases Nonionic and zwitterionic forms of amino acids amphoteric (ampholytes - amphoteric electrolytes ) Titration curves predict the electric charge of amino acids Isoelectric point (or isoelectric pH) pI = ½ (pk 1 + pk 2 ) = ½ (2.34 + 9.60) = 5.97

13. Effect of chemical environment on pK a

14. Amino acids differ in their acid-base properties three stages (three ionization steps  three pk a values) Amino acids with ionizable R groups Amino acids with R groups that do not ionize pk a of the –COOH group: 1.8 – 2.4 pk a of the –NH 3 + group: 8.8 – 11.0 e.g.

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

18. The vast majority of naturally occurring proteins contain fewer than 2,000 amino acid residues. Biologically active peptides and polypeptides occur in a vast range of sizes Multisubunit proteins: have two or more polypeptide chains associated noncovalently 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.

19. Some proteins contain chemical groups other than amino acids (conjugated proteins). Polypeptides have characteristic amino acid compositions 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. 1.0 unit of enzyme activity = amount of enzyme causing the transformation of 1.0  mol of substrate per minute at 25 o C under optimal conditions of measurement. Refers to the total number of units of enzyme in a solution. Number of enzyme units per milligram of total protein. A measure of enzyme purity: it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure.

25. Electrophoresis Cross-linked polymer polyacrylamide acts as a molecular sieve, slowing the migration of proteins approximately in proportion to their charge-to-mass ratio. SDS-polyacrylamide gel SDS CH 3 (CH 2 ) 11 SO 4 - Na + Purification of RNA polymerize from E. coli gel stained with a protein-specific dye (e.g. coomasie blue)

26. Isoelectric focusingTwo-dimensional electrophoresis

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

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 bondsCleaving 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 Correspondence of DNA and amino acid sequences codon

33. Investigating proteins with mass spectrometry

34. Small peptides and proteins can be chemically synthesized

35. (9-fuorenylmethoxycarbonyl)