| Abstract | The ubiquitous calcium-binding protein calmodulin (CaM) regulates a wide variety of cellular events by binding to and activating many distinct target enzymes. The CaM-binding domains of most of these enzymes are contained in a contiguous stretch of amino acids with a length of ≈20 residues. In this work, we have used "isotope-edited" Fourier transform infrared spectroscopy to study the interaction of CaM with synthetic peptides resembling the CaM-binding domains of myosin light chain kinase (MLCK), constitutive nitric oxide synthase (cNOS), and caldesmon (CaD). Uniform labeling of CaM with carbon-13 causes the amide I band of the protein to shift ≈55 cm⁻¹ to lower frequency in D₂0, leaving a clear window in the infrared spectrum for observing the amide I band of the unlabeled target peptides. Upon complex formation, the amide I bands of the CaM-binding domains of MLCK and cNOS shift 4 cm⁻¹ toward higher frequency (to ≈1648 cm⁻¹), and have a narrower bandwidth compared to the peptide in aqueous solution. These spectral changes and the fact that the infrared spectra of these two peptides in their complex with CaM closely resemble those recorded in a mixture of D20 and the helix inducing solvent trifluoroethanol indicate that they bind to CaM in an a-helical conformation. The CaM-binding domain of CaD also showed similar, but less dramatic, spectral changes; this is in agreement with the fact that it binds to CaM with lower affinity and a shorter α-helix. Binding of these three peptides to CaM gave rise to only minor changes in the secondary structure of CaM. The strategy described in this work, which utilizes uniformly isotopically ¹³C-labeled CaM to shift the amide I band of the protein from the original spectral region, should prove useful for determining the secondary structure of CaM-binding domains of other target proteins. Moreover, it should be generally applicable in studies of other protein-protein, protein-peptide, and proteinnucleic acid interactions. |
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