CH339K Proteins: Amino Acids, Primary Structure, and Molecular Evolution

 -Amino Acid 

All amino acids as incorporated are in the L-form All amino acids as incorporated are in the L-form Some amino acids can be changed to D- after incorporation Some amino acids can be changed to D- after incorporation D-amino acids occur in some non-protein molecules D-amino acids occur in some non-protein molecules

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-HydroxyprolineCollagen 5-HydroxylysineCollagen 6-N-MethyllysineHistones  -Carboxygultamate Clotting factors DesmosineElastin SelenocysteineSeveral 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  -amino acid has at least 2 pKa’s

Polymerization  G 0 ’ = kJ/mol  G 0 ’ = kJ/mol

In vivo, amino acids are activated by coupling to tRNA Polymerization of activated a.a.:  G o ’ = kJ/mol

In vitro, a starting amino acid can be coupled to a solid matrix In vitro, a starting amino acid can be coupled to a solid matrix Another amino acid with Another amino acid with A protected amino group A protected amino group An activating group at the carboxy group An activating group at the carboxy group Can be coupled Can be coupled This method runs backwards from in vivo synthesis (C  N) 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 Other amino acids have a trans-cis ratio of ~ 1000:1 Prolines have cis:trans ratio of ~ 3:1 Prolines have cis:trans ratio of ~ 3:1 Ring structure of proline minimizes  G 0 difference Ring structure of proline minimizes  G 0 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 100 Mutations x 100 generations: 1 gets fixed 2 still exist 97 eliminated (most almost immediately)

Rates of Change

Protein Evolution Rates Different proteins have different rates

Rates (cont.) Slow rates in proteins critical to basic functions E.g. histones ≈ 6 x 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 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:

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