Protein Evolution and Analysis February

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

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

Electrophoresis The migration of ions in an electric field F e = qE where q is the charge E is the electric Field strength

Electrophoresis a) F e = qE Opposing this is is the frictional force b) F f = µv where v = velocity of migration µ is the coefficient of friction. Therefore substituting equation a) into b) qE = µv

Electrophoresis qE = µv Therefore when F e = F f v=qE/µ

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.

Paper electrophoresis

Acrylamide gel electrophoresis

Disc gel using a glass tube

Polyacrylamide gel tube Electrophoretogram

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

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

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.

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.

Ion Exchange Chromatography 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 -

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.

Affinity Chromatography

Zonal Ultracentrifugation

Ultracentrifugation Sedimentation

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

Protein Synthesis

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

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 reactions etc.

Amino acid difference matrix for 26 species of cytochrome c Man,chimp0 Rh. monkey1 0 Average differences Horse Donkey cow,sheep dog gray whale rabbit kangaroo Chicken penguin Duck Rattlesnake turtle Bullfrog Tuna fish worm fly silk moth Wheat Bread mold Yeast Candida k 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

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

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.

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

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.

Protein Structure Terminology

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

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.

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)