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Evolution of New Protein Topologies through Multistep Gene Rearrangements. Sergio G Peisajovich , Liat Rockah and Dan S Tawfik. Dibyayan DAS CS 502. Overview. The main issue Protein fold Circular Permutation Sequential Gene rearrangements Active Intermediates.

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evolution of new protein topologies through multistep gene rearrangements

Evolution of New Protein Topologies through Multistep Gene Rearrangements

Sergio G Peisajovich, LiatRockah and Dan S Tawfik

Dibyayan DAS

CS 502

overview
Overview
  • The main issue
  • Protein fold
  • Circular Permutation
  • Sequential Gene rearrangements
  • Active Intermediates
slide3

DNA methyltransferases

A 5-methylcytosine(m5C) class methyltransferase is used – M.HaeIII

3-D structure of the m5C class M.HaeIII

permutation by duplication
Permutation by Duplication

Accounts for evolution of various classes of DNA methyltransferases

P by D mechanism for producing a circular permutant

slide5

N- and C- Terminal

N terminus(-NH2) refers to start of the protein chain

C terminus(-COOH) refers to the end of the protein chain

N - terminus

C - terminus

A tetrapeptide example

slide6

Codons

The coding region of a gene is that portion of the gene’s DNA or RNA that codes for protein

Each set of 3 that codes for an amino acid.

slide7

Creation of Circular Permutants

  • Gene duplication and in frame fusion.
  • Partial degeneration of

5’ coding region of the first copy and

3’ coding region of the second copy of the fused dimer

(ITCHY Methodology)

results
Results
  • N-terminally truncated intermediates was generated by introducing start codons at random locations along the first copy
  • C-terminally truncated intermediates was generated by introducing stop codons at random locations along the second copy
  • C-terminally truncated intermediates were clustered in 3 groups (I, II & III) whereas N-terminally truncated ones in just 2 groups(IV & V)
slide10

DNA Methyltransferases are classified according to the linear order of their conserved motif sequences(I-X) and location of the TRD, defining 7 classes.

Potential new class identified

slide11

Results

  • Intermediates from cluster I and II yieldedband z–class enzymes upon N-terminal truncation
  • Intermediates from cluster IV yielded z-class enzymes upon C-terminal truncation
  • No intermediates lead to g-class enzymes
  • Intermediates with divided TRD were discovered(clusters III and V), were named h
truncated intermediates
TruncatedIntermediates

Do they lead to Circular Permutants?

Yes

How?

By randomly truncating the N-terminally truncated intermediates at their C termini and C-terminally truncated intermediates at their N termini generating different libraries of potential Circular Permutants

results1
Results

Circular Permutants generated by cluster I and IV intermediates

point mutation
Point Mutation
  • Isolate least active intermediate A180-B330
  • Mutate it with circular permutant A44-B61 for M.HaeIII activity
  • Showed markedly enhanced in vitro activity compared to respective starting points
what we found
What we found?
  • Selection either through intermediates or directly for permutants yielded similar results

What it means?

  • In case of DNA methyltransferases, the same structural constraints apply to both the intermediates and the end products
plausibility of p by d model
Plausibility of P by D model
  • Foldability and Functionality of truncated intermediates
  • Do they fold properly? If so, how?
  • What role hydrophobic surfaces play?
  • Hypothesis supported by Power Law
slide18

ANP : Non polar accessible surface area

  • MW : Molecular Weight
  • Power Law : ANP ∝ MW0.73
  • The theoretical dependence of ANP on MW was calculated using :
  • ANP=fNP6.3*MW0.73
  • fNP: nonpolar fraction of the total area of the full length protein
identification of natural h class
Identification of natural h-class
  • No topology has thus far been identified with TRD halved(h-class)
  • Experiments indicated that this topology is functional
  • Bacillus stearothermophilus, E.coli & Hafniaalveiwere identified as members of h-class
conclusion
Conclusion
  • New protein topology can evolve through multistep gene rearrangements
  • Point mutation favors one topology over others
  • Modularity of the methyltransferase fold dictates which intermediates will fold, posses function and eventually yield a new topology
future avenues
Future Avenues
  • Duplicated modules swapped at protein level before permutation is completed at the gene level
  • If other classes, like the h-class, which was first identified in the laboratory, exists?
  • Methods other than Permutation by Duplication