CH339K
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Transcript of CH339K
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CH339K
Proteins: Amino Acids, Primary Structure, and Molecular Evolution
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a-Amino Acid
a
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• 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
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C
HOOC
NH2
R H C
HOOC
NH2
RH
L-amino acid D-amino acid
I prefer this layout, personally…
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2 Amides
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The Acidic and the Amide Amino Acids Exist as Conjugate Pairs
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Ionizable Side Chains
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Hydrogen Bond Donors / Acceptors
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Disulfide formation
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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
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A Modified Amino Acid That Can Kill You
Diphthamide (2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio-propyl)-3H-imidazol-4-yl]propanoate)
Histidine
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• 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
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Corynebacterium diphtheriae Corynebacteriophage
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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
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• Diphtheria toxin adds a bulky group to diphthamide
• eEF2 is inactivated• Cell quits making
protein• Cell(s) die• Victim dies
Diphtheria Toxin Action
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Other Amino Acids
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Every a-amino acid has at least 2 pKa’s
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Polymerization
DG0’ = +10-15 kJ/mol
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In vivo, amino acids are activated by coupling to tRNA
Polymerization of activated a.a.:DGo’ = -15-20 kJ/mol
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• 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)
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Peptide Bond
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Resonance stabilization of peptide bond
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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
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MOLECULAR EVOLUTION
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Time of Divergence|-------------|-------------|------------|------------|-------------|------------| ┌───────────────────────────────Shark │ │ ┌─────────────────────Perch └─────────┤ │ ┌─────────────Alligator └───────┤ │ ┌──────Horse └──────┤ │ ┌───Chimp └──┤ │ └───Human|-------------|-------------|------------|------------|------------|------------|------------|------------|Sequence Difference
Sequence differences among vertebrate hemoglobins
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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
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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)
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Genetic Drift in Action
Ow!
Our green genes are evolutionarily superior!
Never mind…
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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)
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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
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NR
NRNRR
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a
a
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Protein Evolution RatesDifferent proteins have different rates
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Protein Evolution RatesDifferent proteins have different rates
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Rates (cont.)
• Slow rates in proteins critical to basic functions
• E.g. histones ≈ 6 x 10-12 changes/a.a./year
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Rates (cont.)Fibrinopeptides
• Theoretical max mutation rate
• Last step in blood clotting pathway
• Thrombin converts fibrinogen to fibrin
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Fibrinopeptides keep fibrinogens from sticking together.
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Rates (cont.)
• Only constraint on sequence is that it has to physically be there
• Fibrinopeptide limit ≈ 9 x 10-9 changes/a.a./year
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Amino acid sequences of several ribosome-inhibiting proteins
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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
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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