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
C
HOOC
NH2
R H C
HOOC
NH2
RH
L-amino acid D-amino acid
I prefer this layout, personally…
2 Amides
The Acidic and the Amide Amino Acids Exist as Conjugate Pairs
Ionizable Side Chains
Hydrogen Bond Donors / Acceptors
Disulfide formation
4-Hydroxyproline Collagen
5-Hydroxylysine Collagen
6-N-Methyllysine Histones
g-Carboxygultamate Clotting factors
Desmosine Elastin
Selenocysteine Several enzymes (e.g. glutathione peroxidase)
Modified Amino Acids
A Modified Amino Acid That Can Kill You
Diphthamide (2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio-propyl)-3H-imidazol-4-yl]propanoate)
Histidine
• Diphthamide is a modified Histidine residue in Eukaryotic Elongation Factor 2
• EF-2 is required for the translocation step in protein synthesis
Diphthamide Continued – Elongation Factor 2
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 adds a bulky group to diphthamide
• eEF2 is inactivated• Cell quits making
protein• Cell(s) die• Victim dies
Diphtheria Toxin Action
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
Time of Divergence|-------------|-------------|------------|------------|-------------|------------| ┌───────────────────────────────Shark │ │ ┌─────────────────────Perch └─────────┤ │ ┌─────────────Alligator └───────┤ │ ┌──────Horse └──────┤ │ ┌───Chimp └──┤ │ └───Human|-------------|-------------|------------|------------|------------|------------|------------|------------|Sequence Difference
Sequence differences among vertebrate hemoglobins
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
Ow!
Our green genes are evolutionarily superior!
Never mind…
Simulation of Genetic Drift
0 25 50 75 1000
0.2
0.4
0.6
0.8
1
Generation
Freq
uenc
y• 100 Mutations x 100 generations:
• 1 gets fixed• 2 still exist• 97 eliminated (most almost immediately)
Rates of Change
CLOCK MOLECULAR a becan on accumulati change ThereforeCONSTANT. ison accumulati change Therefore
fixation. ofy probabilit theimesmutation t ofy probabilit on theonly depends Rout. cancels size population Therefore
1
size population torelatedboth are and and
ratefixation ratemutation
:where Rate Overall
T
NR
NRNRR
RR
RRR
F
M
FM
F
M
FMT
a
a
Protein Evolution RatesDifferent proteins have different rates
Protein Evolution RatesDifferent 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 RCA-A 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 SNA-IV 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 sequencesIñaki Ruiz-Trillo, Andrew J. Roger, Gertraud Burger, Michael W. Gray & B. Franz Lang (2008) Molecular Biology and Evolution, Jan 9
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