Protein Evolution and Analysis February 5 2003

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

Fe = qE

where q is the charge

E is the electric Field strength

Electrophoresis

a) Fe = qE

Opposing this is is the frictional force

b) Ff = 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 Fe = Ff

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,chimp 0Rh. monkey 1 0 Average differencesHorse 12 11 0Donkey 11 10 1 0 10.0cow,sheep 10 9 3 2 0dog 11 10 6 5 3 0gray whale 10 9 5 4 2 3 0 5.1rabbit 9 8 6 5 4 5 2 0kangaroo 10 11 7 8 6 7 6 6 0Chicken 13 12 11 10 9 10 9 8 12 0penguin 13 12 12 11 10 10 9 8 10 2 0 9.9Duck 11 10 10 9 8 8 7 6 10 3 3 0 14.3Rattlesnake 14 15 22 21 20 21 19 18 21 19 20 17 0 12.6turtle 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 18.5worm 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 0Bread 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 0Candida 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

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)