Nuclear magnetic resonsance studies of side-chain motions in calbindin D(9k): The role of conformational dynamics in protein stability and calcium binding

An accurate understanding of the role of conformational dynamics in proteins requires data at multiple timescales and sites within the protein of interest. Considerable progress has been achieved in characterizing the picosecond-to-nanosecond (ps-ns) dynamics of the protein backbone via NMR relaxation measurements of the 15N nucleus. More recent developments in the measurement of 2H quadrupolar relaxation rates are enabling an extensive characterization of the dynamics in methyl-containing side-chains as well. The aim of the present study is to characterize the effects of Ca2+ binding on the side-chain dynamics of the protein calbindin D 9k. Calbindin is a small (∼8.7 kD), single domain protein of the EF-hand family. It contains two Ca2+ binding sites that exhibit high positive cooperativity. Longitudinal, transverse, quadrupolar order, transverse antiphase and double quantum relaxation rates are reported for both the apo (Ca2+-free) 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 indicate that a methyl group's distance from the Ca2+ binding sites is a significant determinant of 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 achieves high affinity binding while minimizing the corresponding conformational entropy loss.