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Rewiring the Specificity of Two-Component Signal Transduction System

Rewiring the Specificity of Two-Component Signal Transduction System. By Jeffrey M. Skerker , Barrett S. Perchuk , Albert SiryaPorn , Emma A. Lubin , Orr Ashenberg , Mark Goulian , and Michael T. Laub Cell (2008); 133(6); 1043-1054 Presented by Jose R. Echevarria for 20.385 02-24-2010.

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Rewiring the Specificity of Two-Component Signal Transduction System

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  1. Rewiring the Specificity of Two-Component Signal Transduction System By Jeffrey M. Skerker, Barrett S. Perchuk, Albert SiryaPorn, Emma A. Lubin, Orr Ashenberg, Mark Goulian, and Michael T. Laub Cell (2008); 133(6); 1043-1054 Presented by Jose R. Echevarria for 20.385 02-24-2010

  2. Hypothesis Could the Specificity of HistidineKinases be determined by amino acids that exhibit a large degree of covariation. If you vary a subset of amino acids of HistidineKinases, could that modify the specificity of the HistidineKinases.

  3. HistidineKinase Diagram

  4. Structural Analysis based on HistidinePhosphotransferase Residues do not perfectly correlate with contact residues of the Spo0B:Spo0F model

  5. Primary Alignments

  6. Search for covarying residues

  7. Search for Covarying Residues • 43 amino acids found with an MI greater than .35 • 36 of them included the dHp domain of histidinekinase and it’s response regulator • Formula for MI:

  8. DHp Domain is Critical

  9. 7 Residue Chimera Construct Mutants with varying levels of homology to the EnvZDHp domain. Holds 7 Specificity – determining residues at the C- terminal, N-terminal portion of helix 2, and a connecting loop.

  10. More Chimera Testing

  11. Kcat/Km Analysis

  12. Mutation of Residues and Analysis

  13. New Mutants containing Loop

  14. Well behaved Chimeras

  15. In Vivo Testing

  16. Critique Key Assumptions • Phylogeny is greatly reduced by screening for genomes with less than 90 % identity. • Covariance determines the amino acids that would be more deterministic in histidine specificity. Pit-Fall • Need a combination of approaches for better understanding.

  17. Significance • Proof of the possibility to rewire the specificity of input systems by modifying a small number of residue. • Model through which we can add complexity and apply to other biological systems in vivo. • Cross-talk Suppression between the CpxA-CpxR and EnvZ-OmpR Two-Component Systems in E. coli; Albert Siryaporn & Mark Goulian

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