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 Xray 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-) + AR = 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
Rh. monkey
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.
0
1
12
11
10
11
10
9
10
13
13
11
14
15
18
21
27
31
43
48
45
51
0
11
10
9
10
9
8
11
12
12
10
15
14
17
21
26
30
43
47
45
51
Average differences
0
1
3
6
5
6
7
11
12
10
22
11
14
19
22
29
46
46
46
51
0
2
5
4
5
8
10
11
9
21
10
13
18
22
28
45
46
45
50
10.0
0
3
2
4
6
9
10
8
20
9
11
17
22
27
45
46
45
50
0
3
5
7
10
10
8
21
9
12
18
21
25
44
46
45
49
0
2
6
9
9
7
19
8
11
17
22
27
44
46
45
50
5.1
0
6
8
8
6
18
9
11
17
21
26
44
46
45
50
0
12
10
10
21
11
13
18
24
28
47
49
46
51
0
2
3
19
8
11
17
23
28
46
47
46
51
0
3
20
8
12
18
24
27
46
48
45
50
0
17
7
11
17
22
27
46
46
46
51
9.9
14.3
0
12.6
22 0
24 10 0
26 18 15 0
29 24 22 24
31 28 29 32
46 46 48 49
47 49 49 48
47 49 47 47
51 53 51 48
18.5
0
14
45
41
45
47
0
25.9
45 0
47 54 0 47.0
47 47 41 0
47 50 42 27 0
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)