Calmodulin is a ubiquitous calcium signaling protein with a modular and highly flexible structure that allows it to fulfill many regulatory roles associated with calcium signalling. The artificial amino acid cyanylated cysteine is incorporated as a vibrational probe on single-site cysteine mutants of calmodulin. These probe groups are used to determine site-specific conformational distribution and dynamics of calmodulin in different environments. Probe groups were previously incorporated on a calmodulin target peptide, and now several cysteine mutation sites have been chosen across many different possible binding surfaces of calmodulin itself: these mutants have been expressed using e. coli and purified. Infrared spectroscopy is used to show the probe group's local environment in the apo and calcium-saturated states of the free protein as well as bound states with many of calmodulin's possible binding partners. The goal is to map the dynamic and modular binding interfaces through a series of different structures and conformational distributions. Isothermal titration calorimetry and far-UV circular dichroism experiments are performed to determine the extent to which this artificial mutation affects calmodulin's binding affinities and secondary structure, respectively.

Site-specific conformational distributions in calmodulin revealed by cyanylated cysteine infrared probe groups

246th National Meeting (2013)

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