Protein Evolution and Analysis
Protein Assays
• An assay is a method of detection
• Specific
• Sensitive
Enzyme-linked
Immunosorbent
Assay
• Usable in a complex
mixture
Electrophoresis
The migration of ions in an electric field
F
e= qE
where q is the charge
Electrophoresis
a) F
e= qE
Opposing this is is the frictional force
b) F
f= µ
v
where
v
= velocity of migration
µ
is the coefficient of friction.
Therefore substituting equation a) into b)
Electrophoresis
qE = µ
v
Therefore when F
e= F
fSeparates 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
Polyacrylamide
gel tube
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
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
.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.
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.
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
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
Protein Evolution
• Sequence comparisons provide information
on protein structure and function
• Homologous proteins
– Invariant residues
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
.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
Amino acid difference matrix for 26 species of cytochrome c
Man,chimp 0
Rh. monkey 1 0 Average differences
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
PAM values differ for different
proteins.
Although DNA mutates at an
assumed constant rate. Some proteins cannot accept
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
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.
Domains
"Within a single subunit [polypeptide chain],
contiguous portions of the polypeptide chain frequently fold into compact, local semi-independent units called
Mosaic proteins
• Mosaic proteins are those which consist of many repeated
Tertiary Structure and Multi Domains
