Effects of Calcium Binding on the Side-chain Methyl Dynamics of Calbindin D9k: A 2H NMR Relaxation Study

The effects of Ca2+ binding on the side-chain methyl dynamics of calbindin D9k have been characterized by 2H NMR relaxation rate measurements. Longitudinal, transverse in-phase, quadrupolar order, transverse anti-phase and double quantum relaxation rates are reported for both the apo and Ca2+-loaded states of the protein at two magnetic field strengths. The relatively large size of the data set allows for a detailed analysis of the underlying conformational dynamics by spectral density mapping and model-free fitting procedures. The results reveal a correlation between a methyl group's distance from the Ca2+ binding sites and its conformational dynamics. Several methyl groups segregate into two limiting classes, one proximal and the other distal to the binding sites. Methyl groups in these two classes respond differently to Ca2+ binding, both in terms of the timescale and amplitude of their fluctuations. Ca2+ binding elicits a partial immobilization among methyl groups in the proximal class, which is consistent with previous studies of calbindin's backbone dynamics. The distal class, however, exhibits a trend that could not be inferred from the backbone data in that its mobility actually increases with Ca2+ binding. We have introduced the term polar dynamics to describe this type of organization across the molecule. The trend may represent an important mechanism by which calbindin D9k achieves high affinity binding while minimizing the corresponding loss of conformational entropy.