Site-Specific Conformational Diversity in Calmodulin using Cyanylated Cysteine Vibrational Probes along its Binding Interfaces

Abstract

Vibrational spectroscopy was used to probe the modular binding interfaces of calmodulin. Two hydrophobic amino acid patches allow calmodulin great flexibility in binding to target partners, and these patches contain key methionine residues. Site-specific mutagenesis was used to generate numerous single cysteine variants of calmodulin, including three mutations at key methionine residues, which were expressed, purified and cyanylated at cysteine to generate a vibrational probe group at each site. Infrared spectroscopy was used to assess the environment surrounding each probe in apo- and calcium saturated conditions, as well as complexed with the binding peptide from skeletal muscle myosin light chain kinase. Circular dichroism (CD) experiments were conducted to ensure the SCN vibrational probe did not perturb calmodulin's secondary structure, and isothermal calorimetry was performed for each labeled variant with the skMLCK peptide to ensure the probe did not disrupt complex formation. Surprisingly, the methionine to cyanylated cysteine mutation at key methionine residues did not lead to a large perturbation in binding thermodynamics. This suggests that the cyanylated cysteine probe group is relatively innocent even when placed directly in the binding interface and can report directly on structural dynamics along the binding interface between calmodulin and its many targets.