Ultrafast two-dimensional infrared (2DIR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Ca2+ and several trivalent lanthanide ions) and the ion-coordinating sites of the calmodulin (CaM) calcium-sensing protein. Using a series of lanthanide ions allowed us to finely tune structural distortion of CaM's binding site with a degree of control far greater than is possible with other methods such as site-directed mutagenesis. CaM is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to subtle patterns of Ca2+ exposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Despite extensive study, many details of this transduction function are not well understood. Although functional assays indicate that Tb3+ fully activates many Ca2+-dependent proteins, our FTIR spectra indicate that Tb3+, La3+, and Lu3+ disrupt the unique bidentate coordination geometry characteristic of the CaM binding sites’ strongly conserved position 12 glutamate residue. 2DIR spectra indicate that, relative to the lanthanide-bound forms, Ca2+-bound CaM maximizes vibrational energy transfer between carboxylate and carbonyl groups in the protein, consistent with subtle conformational differences between these species. The results demonstrate that FTIR and 2DIR are sensitive to aspects of ion binding to CaM that remain invisible to other biophysical methods.

Probing ion-dependent changes to calmodulin binding site conformation with FTIR and ultrafast 2DIR spectroscopy

255th National Meeting (2018)

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