Journal of Proteomics & Bioinformatics
Open Access

Engineering a “metal switch” into molecular motors to control their activity

International Conference on Protein Engineering

October 26-28, 2015 Chicago, USA

Jared C Cochran

Indiana University, USA

Posters-Accepted Abstracts: J Proteomics Bioinform

Abstract :

Kinesins and myosins are molecular motors that use the energy from nucleotide hydrolysis to carry out cellular tasks. In addition to the P-loop, these proteins use similar structural motifs called switch-1 and switch-2, to sense and respond to the gammaphosphate of the nucleotides and coordinate nucleotide hydrolysis. We have developed a strategy to probe metal interactions within kinesins and myosins by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine (-OH) and cysteine (-SH) amino acids. We present the crystal structure of a recombinant kinesin motor domain bound to MnADP and report on a serine-tocysteine substitution in the switch-1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn(II) to Mg(II). This mutant kinesin binds ATP similarly in the presence of either metal ion but its ATP hydrolysis activity is greatly diminished in the presence of Mg(II). In multiple kinesin members, this defect is rescued by Mn(II), providing a way to control both the enzymatic activity and force-generating ability of these nanomachines. We also present results for an analogous substitution in non-muscle myosin-2. This mutant myosin shows aberrant actin interaction whereby dissociation becomes rate-limiting in the presence of Mg(II), yet is rescued by Mn(II). There are several relevant and important applications to this metal switch technology that will allow further biophysical characterization of molecular motors and molecular switch proteins.

Biography :

Email: jcc6@indiana.edu