1. Ch.5 Proteins: Primary structure Polypeptide diversity Protein purification and analysis Protein sequencing Protein evolution

2. Polypeptide diversity Primary structure = the amino acid sequence of its polypeptide chain(s)  What is the importance of primary structure?  Are there limits to the size and composition of naturally occurring proteins? Ch.5 Proteins: Primary structure A representation of the primary structure of insulin (bovine)

3. Polypeptide diversityCh.5 Proteins: Primary structure

4. Protein purification and analysis Purifying a protein requires a strategy Salting out separates proteins by their solubility Chromatography involves interaction with mobile and stationary phases Electrophoresis separates molecules according to charge and size Ch.5 Proteins: Primary structure

5. Purifying a protein requires a strategy Source Conditions such as pH, temperature, ionic strength must be controlled and optimized Assay of protein activity Spectrophotometric and other methods used for determination of concentration Purification is a stepwise process Ch.5 Proteins: Primary structure

6. Purifying a protein requires a strategy Ch.5 Proteins: Primary structure

7. Salting out separates proteins by their solubility Protein solubility generally increases with increasing ionic strength, reaches a maximum, then decreases with further increase in ionic strength Ammonium sulfate is the ionic compound of choice for salting out proteins Ch.5 Proteins: Primary structure

8. Chromatography involves interaction with mobile and stationary phases Ion exchange Hydrophobic interaction Gel filtration Affinity Ch.5 Proteins: Primary structure The most common types of chromatography

9. Electrophoresis separates molecules according to charge and size SDS-PAGE Capillary electrophoresis 2D-electrophoresis Ch.5 Proteins: Primary structure

10. pI, pH, acidic & basic proteins The pI, or isoelectric point, of a protein is the pH at which the net charge on the molecule is zero If pH > pI, then the protein is negatively charged (acidic proteins have pI < 7) If pH 7)

11. Charge vs. pH for a protein

12. Protein sequencing Separate subunits; (identification of N-terminal residues) Cleave polypeptide chains (using at least two methods to generate distinct sets of peptide fragments) Sequence fragments  Chemically (by Edman degradation)  Mass spectrometry Assemble sequence data to yield primary structure Ch.5 Proteins: Primary structure

13. Protein sequencing Ch.5 Proteins: Primary structure See Problems 14-18

14. Generation of peptide fragments: Endopeptidases Trypsin  Cleaves on the C-terminal side of Arg or Lys residues  High specificity; does not cleave if following residue is Pro Chymotrypsin  Cleaves on the C-terminal side of Phe, Trp, Tyr  Less specificity; does not cleave if following residue is Pro See Table 5-3, p.107

15. Generation of peptide fragments: Chemical methods Cyanogen bromide (CNBr)  Cleaves on the C-terminal side of Met residues  Generates an N-terminal fragment that is a peptidyl homoserine lactone

16. Protein sequencing by Edman degradation Ch.5 Proteins: Primary structure A three-stage reaction that labels and removes the N-terminal residue of a polypeptide Reaction can be run repetitively to sequence up to 100 residues in favorable cases Automated in modern instrumentation and can be performed on small amounts of a peptide (5-10 pmol or <0.1μg

17. Protein sequencing by Edman degradation Ch.5 Proteins: Primary structure Problem: Method requires a free N-terminus, and therefore polypeptides with blocked N-termini (e.g. acetylation) cannot be sequenced by Edman degradation Despite automation, the process takes a considerable amount of time

18. Protein sequencing by mass spectrometry Ch.5 Proteins: Primary structure Tandem mass spectrometry (MS/MS) can be used to sequence short peptides (<25 residues)

19. Protein sequencing by mass spectrometry Ch.5 Proteins: Primary structure Advantages:  Method can deal with blocked N-termini  Rapid acquisition of sequence data  Enables characterization of common post-translational modifications

20. Protein sequencing by mass spectrometry Ch.5 Proteins: Primary structure Limitation: Inability to distinguish Ile and Leu, difficulty in distinguishing Gln and Lys

21. Mass spectrometry of proteins Ch.5 Proteins: Primary structure Mass spectrometry measures mass-to-charge ratio (m/z) for ions in gas phase Electrospray ionization (ESI) mass spectrometry is an accurate method for determining mass of intact polypeptides

22. Protein sequence analysis and databases Ch.5 Proteins: Primary structure

23. Protein sequence analysis and databases Ch.5 Proteins: Primary structure NCBI: http://www.ncbi.nlm.nih.gov/BLAST

24. Protein evolution Ch.5 Proteins: Primary structure Protein sequences reveal evolutionary relationships Proteins evolve by the duplication of genes or gene segments  Protein families can arise through gene duplication e.g. globin family  The rate of sequence divergence varies  Many proteins contain domains that occur in other proteins

25. Protein evolution Ch.5 Proteins: Primary structure Terms to be familiar with:  Homologous proteins Distinguish between orthologous and paralogous. Domains  With respect to residues in multiple sequence alignments: Invariant Conservative substitutions Hypervariable positions

26. Protein evolution Ch.5 Proteins: Primary structure

27. Protein evolution Ch.5 Proteins: Primary structure

28. Protein evolution Ch.5 Proteins: Primary structure

29. Protein evolution Ch.5 Proteins: Primary structure