4.4 Article

Non-Additive Effects of Binding Site Mutations in Calmodulin

Journal

BIOCHEMISTRY
Volume 58, Issue 24, Pages 2730-2739

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.9b00096

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Funding

  1. College of Natural Sciences at The University of Texas at Austin
  2. Welch Foundation [F-1891]
  3. National Institutes of Health [R01NS077821]
  4. National Science Foundation [BIO-1815354]

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Despite decades of research on ion-sensing proteins, gaps persist in the understanding of ion binding affinity and selectivity even in well-studied proteins such as calmodulin. Site-directed mutagenesis is a powerful and popular tool for addressing outstanding questions about biological ion binding and is employed to selectively deactivate binding sites and insert chromophores at advantageous positions within ion binding structures. However, even apparently nonperturbative mutations can distort the binding dynamics they are employed to measure. We use Fourier transform infrared (FTIR) and ultrafast two-dimensional infrared (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 two classes of mutation, tryptophan insertion and carboxylate side-chain deletion, commonly used to study ion binding in proteins. Our results show that these mutations not only affect ion binding 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 line shapes with 2D IR diagonal slices provides a clear example of how nonlinear spectroscopy produces well-resolved line shapes, refining otherwise featureless spectral envelopes into more informative vibrational spectra of proteins.

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