Phi Analysis of CusF: Alanine-scanning Mutagenesis to Reveal the Folding Transition State

Authors:

Alina Mitina, James Wondra

Mentor:

Blake Gillespie, Professor of Chemistry, CSU Channel Islands

What are the key interactions of protein structure that govern their folding kinetics? Can the presence of ligand affect the folding process? We are using the copper chaperone protein CusF as a model system for the exploration of folding pathways, employing a protein engineering strategy in which each amino acid is systematically substituted with alanine. Each mutant is scored for the effects of the substitution on the protein’s equilibrium stability and folding kinetics. A score (or phi-value) of 1 indicates that the structure an amino acid populates in the folded state is also formed in the folding transitions state, whereas a phi-value of 0 indicates that the structure is not formed in the transition state. In this way, we are building a map of the interactions critical to CusF’s folding reaction. So far, we have mutated some 92% of CusF’s sequence. We report a preliminary analysis of a subset of mutants, and we compare our crude map to the transition states of other proteins. None of the 7 mutants thus far characterized have phi-values of 1, indicating that either 1) we have not identified transition state structures or 2) CusF’s transition state is not well-defined by native-like interactions. Future work will focus the characterization of the other 68 alanine mutants. Interestingly, one mutant (P38A) appears to fold much faster than wildtype. The possibility that P38 is involved in a cis-trans isomerization reaction that slows wild-type CusF’s observed refolding rate requires a more careful analysis of our mutant folding kinetics. We are currently investigating whether proline isomerization is important to CusF folding reaction.


Presented by:

James Wondra, Alina Mitina

Date:

Saturday, November 23, 2013

Poster:

104

Room:

Poster Session 1 - Villalobos Hall

Presentation Type:

Poster Presentation

Discipline:

Biochemistry