Centrin is a calcium binding protein (CaBP) belonging to the EF-hand superfamily. As with other proteins within this family, centrin is a calcium sensor with multiple biological target proteins. We chose to study Chlamydomonas reinhardtii centrin (Crcen) and its interaction with melittin (MLT) as a model for CaBP complexes due to its amphipathic properties. Our goal was to determine the molecular interactions that lead to centrin–MLT complex formation, their relative stability, and the conformational changes associated with the interaction, when compared to the single components. For this, we determined the thermodynamic parameters that define Crcen–MLT complex formation. Two-dimensional infrared (2D IR) correlation spectroscopy were used to study the amide I′, I′*, and side chain bands for 13C-Crcen, MLT, and the 13C-Crcen–MLT complex. This approach resulted in the determination of MLT's increased helicity, while centrin was stabilized within the complex. Herein we provide the first complete molecular description of centrin–MLT complex formation and the dissociation process. Also, discussed is the first structure of a CaBP–MLT complex by X-ray crystallography, which shows that MLT has a different binding orientation than previously characterized centrin-bound peptides. Finally, all of the experimental results presented herein are consistent with centrin maintaining an extended conformation while interacting with MLT. The molecular implications of these results are: (1) the recognition of hydrophobic contacts as requirements for initial binding, (2) minimum electrostatic interactions within the C-terminal end of the peptide, and (3) van der Waals interactions within MLTs N-terminal end are required for complex formation. Proteins 2011; © 2011 Wiley-Liss, Inc.
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