CH339K

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