We use FTIR and ultrafast 2D IR spectroscopy of the carboxylate asymmetric stretching mode in calmodulin as a mutation- and label-independent probe of the conformational perturbations induced in calmodulin's binding sites by different classes of site-directed mutation commonly used to study ion binding in proteins.<br/><br/>Site-directed mutagenesis is a popular tool for addressing outstanding questions about biological ion binding. However, mutagenesis can have unintended structural effects, and even apparently non-perturbative mutations can distort the binding dynamics they are employed to measure. Due to their picometer spatial resolution, sub-picosecond temporal resolution, and direct connection to molecular structure, the vibrational spectroscopies we employ are well-positioned to interrogate mutation-dependent characteristics of binding site structure and dynamics.<br/><br/>Results show that the mutations under study not only affect ion binding in unexpected ways, but also induce changes in calmodulin's conformational landscape along coordinates not probed by vibrational spectroscopy, remaining invisible without additional perturbation of binding site structure. Comparison of FTIR lineshapes with 2D IR data provide a clear example of how nonlinear spectroscopy can provide exceptionally informative vibrational spectra of proteins.

Ultrafast vibrational spectroscopy reveals non-additive effects of mutation on binding site structure

2019 Spring National Meeting

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