Protein Evolution and Analysis February 5 2003

1. Protein Evolution and AnalysisFebruary 5 2003

2. Protein Assays • An assay is a method of detection • Specific • Sensitive • Convenient to use

3. Enzyme-linked Immunosorbent Assay • Usable in a complex mixture • High sensitivity

4. Electrophoresis The migration of ions in an electric field Fe = qE where q is the charge E is the electric Field strength

5. Electrophoresis a) Fe = qE Opposing this is is the frictional force b) Ff = µv where v = velocity of migration µis the coefficient of friction. Therefore substituting equation a) into b) qE = µv

6. Electrophoresis qE = µv Therefore when Fe = Ff v=qE/µ

7. Separates on charge and size pH matters as well as the pI of the protein. Can be run at several pH values depending on proteins. DNA can also be separated on agarose gels. Genomic sized DNA can also be separated but requires more sophisticated equipment.

8. Paper electrophoresis

9. Acrylamide gel electrophoresis

10. Disc gel using a glass tube

11. Polyacrylamide gel tube Electrophoretogram

12. Proteins can be visualized by several methods Stained with a Dye: Coomassie blue Fluorescamine stain for fluorescence Silver staining very sensitive proteins can be labeled with radioactivity and visualized by exposure to X- ray film

13. SDS-PAGE Add sodium dodecyl sulfate, a 12 carbon detergent to give a negative charge to the protein. SDS also denatures the protein and collapses into a globular ball. The proteins are separated by molecular mass

15. Chromatography • Analytical methods used to separate molecules. Involves a mobile and a stationary phase. • Mobile phase is what the material to be separated is dissolved in. • Stationary phase is a porous solid matrix which the mobile phase surrounds. • Separation occurs because of the differing chemistries each molecule has with both the mobile and stationary phase. • Chemistries are different depending on the specific method.

16. Types of chromatography • Gas - liquid: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. • Liquid - Liquid: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. • If separation is based on ionic interaction the method is called Ion Exchange chromatography. • If separation is based on solubility differences between the phases the method is called adsorption chromatography. • If the separation is base on size of molecule the method is called gel filtration or size exclusion. • If the separation is base on ligand affinity the method is called Affinity chromatography.

17. Ion ExchangeChromatography • A solid matrix with a positive charge i.e. R+ can bind different anions with different affinities. • We can swap one counter ion for another • (R+A-) + B- (R+B-) + A- • R = Resin and exchanges Anions (-) • This is an anion exchange resin. • There are also cation exchange resins. The type of an R group can determine the strength of interaction between the matrix, R and the counter ion. • If R is R- • (R-A+) + B+ (R-B+) + A-

18. Proteins have a net charge. The charge is positive below pI, while the charge is negative above pI The choice of exchange resin depends on the charge of the protein and the pH at which you want to do the purification. Once the protein binds, all unbound proteins are washed off the column. Bound proteins are eluted by increasing the ionic strength, changing the counter ion or changing the pH altering the charge on the protein or the column.

21. Affinity Chromatography

22. Zonal Ultracentrifugation

23. Ultracentrifugation Sedimentation

24. Protein Evolution • Sequence comparisons provide information on protein structure and function • Homologous proteins • Invariant residues • Conservatively substituted • Hypervariable

25. Protein Synthesis

27. Species variation in homologous proteins The primary structures of a given protein from related species closely resemble one another. If one assumes, according to evolutionary theory, that related species have evolved from a common ancestor, it follows that each of their proteins must have likewise evolved from the corresponding ancestor. A protein that is well adapted to its function, that is, one that is not subject to significant physiological improvement, nevertheless continues to evolve. Neutral drift: changes not effecting function

28. Homologous proteins (evolutionarily related proteins) Compare protein sequences: Conserved residues, i.e invariant residues reflect chemical necessities. Conserved substitutions, substitutions with similar chemical properties Asp for Glu, Lys for Arg, Ile for Val Variable regions, no requirement for chemical reactionsetc.

29. Amino acid difference matrix for 26 species of cytochrome c Man,chimp0 Rh. monkey 1 0 Average differences Horse 12 11 0 Donkey 11 10 1 0 10.0 cow,sheep 10 9 3 2 0 dog 11 10 6 5 3 0 gray whale 10 9 5 4 2 3 0 5.1 rabbit 9 8 6 5 4 5 2 0 kangaroo 10 11 7 8 6 7 6 6 0 Chicken 13 12 11 10 9 10 9 8 12 0 penguin 13 12 12 11 10 10 9 8 10 2 0 9.9 Duck 11 10 10 9 8 8 7 6 10 3 3 0 14.3 Rattlesnake 14 15 22 21 20 21 19 18 21 19 20 17 0 12.6 turtle 15 14 11 10 9 9 8 9 11 8 8 7 22 0 Bullfrog 18 17 14 13 11 12 11 11 13 11 12 11 24 10 0 Tuna fish 21 21 19 18 17 18 17 17 18 17 18 17 26 18 15 0 18.5 worm fly 27 26 22 22 22 21 22 21 24 23 24 22 29 24 22 24 0 silk moth 31 30 29 28 27 25 27 26 28 28 27 27 31 28 29 32 14 0 25.9 Wheat 43 43 46 45 45 44 44 44 47 46 46 46 46 46 48 49 45 45 0 Bread mold 48 47 46 46 46 46 46 46 49 47 48 46 47 49 49 48 41 47 54 0 47.0 Yeast 45 45 46 45 45 45 45 45 46 46 45 46 47 49 47 47 45 47 47 41 0 Candida k. 51 51 51 50 50 49 50 50 51 51 50 51 51 53 51 48 47 47 50 42 27 0 Man,chimp monkey Horse Donkey cow,sheep dog gray whale rabbit kangaroo Chicken, penguin Duck Rattlesnake turtle Bullfrog Tuna fish worm fly silkworm Wheat Bread mold Yeast Candida

30. Phylogenetic tree Indicates the ancestral relationships among the organisms that produced the protein. Each branch point indicates a common ancestor. Relative evolutionary distances between neighboring branch points are expressed as the number of amino acid differences per 100 residues of the protein. PAM units or Percentage of Accepted Mutations

32. PAM values differ for different proteins. Although DNA mutates at an assumed constant rate. Some proteins cannot accept mutations because the mutations kill the function of the protein and thus are not viable.

33. Mutation rates appear constant in time Although insects have shorter generation times than mammals and many more rounds of replication, the number of mutations appear to be independent of the number of generations but dependent upon time Cytochrome c amino acid differences between mammals, insects and plants note the similar distances

34. Evolution through gene duplication • Many proteins within an organism have sequence similarities with other proteins. • These are called gene or protein families. • The relatedness among members of a family can vary greatly. • These families arise by gene duplication. • Once duplicated, individual genes can mutate into separate genes. • Duplicated genes may vary in their chemical properties due to mutations. • These duplicate genes evolve with different properties. • Example the globin family.

35. Protein Structure Terminology

36. Domains "Within a single subunit [polypeptide chain], contiguous portions of the polypeptide chain frequently fold into compact, local semi-independent units called domains." - Richardson, 1981

37. Mosaic proteins • Mosaic proteins are those which consist of many repeated copies of one or a few domains, all within one polypeptide chain. The domains in question are termed modules and are sometimes relatively small.

38. Tertiary Structure and Multi Domains • The domain can perhaps be considered the unit of tertiary structure (c.f. helices and sheets, the units of secondary structure)