CH339K
Proteins: Amino Acids, Primary Structure, and
Molecular Evolution
a-Amino Acid
a
•All amino acids as incorporated are in the L-form
• Some amino acids can be changed to D- after
incorporation
• D-amino acids occur in some non-protein molecules
I prefer this layout, personally…
HOOC
R C H
NH2
D-amino acid
HOOC
H
C
R
NH2
L-amino acid
2 Amides
The Acidic and the Amide Amino Acids Exist
as Conjugate Pairs
Ionizable Side Chains
Hydrogen Bond Donors / Acceptors
Disulfide formation
Modified Amino Acids
4-Hydroxyproline
Collagen
5-Hydroxylysine
Collagen
6-N-Methyllysine
Histones
g-Carboxygultamate
Clotting factors
Desmosine
Elastin
Selenocysteine
Several enzymes (e.g. glutathione peroxidase)
A Modified Amino Acid That Can Kill You
Histidine
Diphthamide
(2-Amino-3-[2-(3-carbamoyl-3-trimethylammoniopropyl)-3H-imidazol-4-yl]propanoate)
Diphthamide Continued – Elongation Factor 2
• Diphthamide is a modified
Histidine residue in
Eukaryotic Elongation
Factor 2
• EF-2 is required for the
translocation step in protein
synthesis
Corynebacterium diphtheriae
Corynebacteriophage
Diphtheria Toxin Action
• Virus infects bacterium
• Infected bacxterium
produces toxin
• Toxin binds receptor on
cell
• Receptor-toxin complex
is endocytosed
• Endocytic vessel
becomes acidic
• Receptor releases toxin
• Toxin escapes
endocytic vessel into
cytoplasm
• Bad things happen
Diphtheria Toxin Action
• Diphtheria toxin
adds a bulky group
to diphthamide
• eEF2 is inactivated
• Cell quits making
protein
• Cell(s) die
• Victim dies
Other Amino Acids
Every a-amino acid has at
least 2 pKa’s
Polymerization
DG0’ = +10-15 kJ/mol
In vivo, amino acids are
activated by coupling to
tRNA
Polymerization of activated
a.a.:
DGo’ = -15-20 kJ/mol
• In vitro, a starting amino acid
can be coupled to a solid matrix
• Another amino acid with
• A protected amino group
• An activating group at the
carboxy group
• Can be coupled
• This method runs backwards
from in vivo synthesis (C N)
Peptide Bond
Resonance stabilization of
peptide bond
Cis-trans isomerization in prolines
•Other amino acids have a trans-cis ratio of ~ 1000:1
•Prolines have cis:trans ratio of ~ 3:1
•Ring structure of proline minimizes DG0 difference
MOLECULAR EVOLUTION
Sequence differences among vertebrate
hemoglobins
Time of Divergence
|-------------|-------------|------------|------------|-------------|------------|
┌───────────────────────────────Shark
│
│
┌─────────────────────Perch
└─────────┤
│
┌─────────────Alligator
└───────┤
│
┌──────Horse
└──────┤
│ ┌───Chimp
└──┤
│
└───Human
|-------------|-------------|------------|------------|------------|------------|------------|-----------|
Sequence Difference
Neutral Theory of Molecular Evolution
• Kimura (1968)
• Mutations can be:
– Advantageous
– Detrimental
– Neutral (no good or bad phenotypic effect)
• Advantageous mutations are rapidly
fixed, but really rare
• Diadvantageous mutations are rapidly
eliminated
• Neutral mutations accumulate
What Happens to a Neutral
Mutation?
• Frequency subject to random chance
• Will carrier of gene reproduce?
• Many born but few survive
– Partly selection
– Mostly dumb luck
• Gene can have two fates
– Elimination (frequent
– Fixation (rare)
Genetic Drift in Action
Our green
genes are
evolutionarily
superior!
Ow!
Never
mind…
Simulation of Genetic Drift
• 100 Mutations x 100 generations:
• 1 gets fixed
• 2 still exist
• 97 eliminated (most almost immediately)
1
Frequency
0.8
0.6
0.4
0.2
0
0
25
50
Generation
75
100
Rates of Change
OverallRate RT  RM  RF
where :
RM  mutationrate
RF  fixationrate
and RM and RF are both relatedto populationsize N
RM a N
1
N
T hereforepopulationsize cancelsout.
R T depends only on theprobability of mutation imes
t
theprobability of fixation.
RF a
T hereforechangeaccumulation is CONST ANT .
T hereforechangeaccumulation can be a MOLECULARCLOCK
Protein Evolution Rates
Different proteins have different rates
Protein Evolution Rates
Different proteins have different rates
Rates (cont.)
• Slow rates in proteins critical to basic
functions
• E.g. histones ≈ 6 x 10-12
changes/a.a./year
Rates (cont.)
Fibrinopeptides
• Theoretical max
mutation rate
• Last step in blood
clotting pathway
• Thrombin converts
fibrinogen to fibrin
Fibrinopeptides keep fibrinogens from sticking together.
Rates (cont.)
• Only constraint on sequence is that it has to
physically be there
• Fibrinopeptide limit ≈ 9 x 10-9 changes/a.a./year
Amino acid sequences of
several ribosome-inhibiting
proteins
Phylogenetic trees built from
the amino acid sequences of
type 1 RIP or A chains (A)
and B chains (B) of type 2
RIP (ricin-A, ricin-B, and lectin RCAA and RCA-B from castor bean;
abrin-A, abrina/b-B, and agglutinin
APA-A and APA-B from A.
precatorius; SNAI-A and SNAI-B,
SNAV-A and SNAV-B, SNAI'-A and
SNAI'-B, LRPSN1-A and LRPSN1-B,
LRPSN2-A and LRPSN2-B, and SNAIV from S. nigra; sieboldinb-A,
sieboldinb-B, SSAI-A, and SSAI-B
from S. sieboldiana; momordin and
momorcharin from Momordica
charantia; MIRJA from Mirabilis
jalapa; PMRIPm-A and PMRIPm-B,
PMRIPt-A and PMRIPt-B from
Polygonatum multiflorum;
RIPIriHol.A1, RIPIriHol.A2, and
RIPIriHol.A3 from iris hybrid; IRAr-A
and IRAr-B, IRAb-A and IRAb-B from
iris hybrid; SAPOF from S.
officinalis; luffin-A and luffin-B from
Luffa cylindrica; and karasurin and
trichosanthin from Trichosanthes
kirilowii)
Hao Q. et.al. Plant Physiol. 2010:125:866-876
Phylogenetic tree of Opisthokonts, based on nuclear
protein sequences
Iñaki Ruiz-Trillo, Andrew J. Roger, Gertraud Burger, Michael W. Gray & B. Franz
Lang (2008) Molecular Biology and Evolution, Jan 9