The Mayo Lab | lab members

Jennifer Keeffe
1200 E. California Blvd.
Pasadena, CA 91125-9600
Mail Code: 114-96

Location: 130 Broad
(626) 395-6407

Research

Designing calmodulin specificity: Incorporation of explicit negative design
Calmodulin (CaM) is an ideal system for studying the specificity of protein-protein interactions by computational design. CaM is a small Ca2+-binding protein that binds to and regulates numerous proteins in vivo with high affinity, including smooth muscle myosin light chain kinase (smMLCK) and CaM kinase I (CaMKI). In addition, several high-resolution CaM-peptide complexes have been solved by X-ray crystallography.

Shifman and Mayo [1] successfully used the ORBIT computational design software to generate a CaM variant with increased specificity toward the smMLCK peptide. However, this feat was accomplished by optimizing the interface between CaM and the smMLCK peptide and did not use any negative design to disfavor the binding of other CaM-binding peptides. In addition, this variant did not discriminate between the smMLCK and CaMKI peptides [2], indicating that there is potential for improvement by including a negative design component against the CaMKI target.

Our current studies are focused on designing a CaM variant that can efficiently discriminate between the smMLCK peptide and the CaMKI peptide by incorporating explicit negative design. Toward this goal, we are using ORBIT to optimize 25 CaM positions in the peptide-binding interface. Using an optimization algorithm written by graduate student Benjamin Allen, the positions are simultaneously optimized on the CaM-smMLCK peptide structure (for a favorable solution) and the CaM-CaMKI peptide structure (for an unfavorable solution). We are currently investigating various negative design scoring functions and parameters to obtain the most favorable results. Future experimental validation of the computationally designed variants will include tryptophan fluorescence titrations to determine the binding affinity of CaM to the peptides.