Protein Evolution and Analysis February 5 2003

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Protein Evolution and Analysis February 5 2003
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Protein Assays

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

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

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Electrophoresis
The migration of ions in an electric field
Fe qE where q is the charge E is the electr
ic Field strength

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Electrophoresis
a) Fe qE Opposing this is is the frictional for
ce b) Ff µv where v velocity of migrati
on µ is the coefficient of friction. Therefore
substituting equation a) into b) qE µv
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Electrophoresis
qE µv Therefore when Fe Ff vqE/µ
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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 ge
ls. Genomic sized DNA can also be separated but
requires more sophisticated equipment.
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Paper electrophoresis
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Acrylamide gel electrophoresis
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Disc gel using a glass tube
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Polyacrylamide gel tube Electrophoretogram
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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 t
o X- ray film
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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
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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.

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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.

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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
• (RA-) B- ? (RB-) 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-

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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 char
ge 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.
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Affinity Chromatography
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Zonal Ultracentrifugation
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Ultracentrifugation Sedimentation
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Protein Evolution

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

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Protein Synthesis
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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
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Homologous proteins (evolutionarily related prot
eins)
Compare protein sequences Conserved residues, i.
e invariant residues reflect chemical
necessities. Conserved substitutions, substitut
ions with similar chemical properties Asp for
Glu, Lys for Arg, Ile for Val Variable regions, n
o requirement for chemical reactions etc.
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Amino acid difference matrix for 26 species of
cytochrome c
Man,chimp 0 Rh. monkey 1 0 Average d
ifferences 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 1
0 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 1
3 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 2
8 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 4
6 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 Tu
na fish worm fly silkworm Wheat Bread mold
Yeast Candida
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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 Acc
epted Mutations
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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.
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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
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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.

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Protein Structure Terminology
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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

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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.

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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)