Structural dynamics in the C-terminal domain of calmodulin at low calcium levels

Calmodulin undergoes Ca2+-induced structural rearrangements that are intima tely coupled to the regulation of numerous cellular processes. The C-termin al domain of calmodulin has previously been observed to exhibit conformatio nal exchange in the absence of Ca2+. Here, we characterize further the conf ormational dynamics in the presence of low concentrations of Ca2+ using N-1 5 spin relaxation experiments. The analysis included H-1-N-15 dipolar/N-15 chemical shift anisotropy interference crosscorrelation relaxation rates to improve the description of the exchange processes, as well as the picoseco nd to nanosecond dynamics. Conformational transitions on microsecond to mil lisecond time stales were revealed by exchange contributions to the transve rse auto-relaxation rates. In order to separate the effects of Ca2+ exchang e from intramolecular conformational exchange processes in the apo state, t ransverse auto-relaxation rates were measured at different concentrations o f free Ca2+ The results reveal a Ca2+-dependent contribution due mainly to exchange between the apo and (Ca2+), states with an apparent Ca2+ off-rate of similar to 5115 s(-1), as well as Ca2+-independent contributions due to conformational exchange within the apo state. N-15 chemical shift differenc es estimated from the exchange data suggest that the first Ca2+ binds prefe rentially to loop IV. Thus, characterization of chemical exchange as a func tion of Ca2+ concentration has enabled the extraction of unique information on the rapidly exchanging and weakly populated (<10%) (Ca2+)(1) state that is otherwise inaccessible to direct study due to strongly cooperative Ca2 binding. The conformational exchange within the apo state appears to invol ve transitions between a predominantly populated closed conformation and a smaller population of more open conformations. The picosecond to nanosecond dynamics of the apo state are typical of a well-folded protein, with reduc ed amplitudes of motions in the helical segments, but with significant flex ibility in the Ca2+-binding loops. Comparisons with order parameters for sk eletal troponin C and calbindin D-9k reveal key structural and dynamical di fferences that correlate with the different Ca2+-binding properties of thes e proteins. (C) 1999 Academic Press.