NMR studies on calcium-induced conformational transitions in calmodulin

Abstract: Nuclear magnetic resonance (NMR) spectroscopy was used in order to investigate the relationships between structure, dynamics and calcium binding in the intracellular regulatory protein, calmodulin. Calmodulin consists of two similar domains, each binding two calcium ions with positive cooperativity. The studies described in this thesis focus on the isolated C-terminal domain, TR2C, and two mutants thereof. TR2C consists of two calcium-binding helix- loop-helix motifs packed together in a parallel fashion. The three-dimensional solution structures of TR2C were determined with and without calcium ions bound.The two structures are similar in terms of secondary structure, but large reorientations of the helices occur upon calcium binding, yielding an open conformation with a large exposed hydrophobic surface. Furthermore, the structures of the calcium- free and calcium-loaded states of TR2C were shown to be highly similar to those of the corresponding domain of intact calmodulin, demonstrating the structural autonomy of the domains. In each of the two mutants, E104Q and E140Q, a conserved bidentate calcium-coordinating glutamic acid residue has been mutated to a glutamine in one of the two binding sites. Calcium titration data were obtained from NMR spectra and the binding constants were determined. Both mutants bind calcium sequentially, but in the opposite order, and the structural response was found to be different for the two calcium-binding sites. The calcium-free states of the mutants are very similar to the calcium-free state of wild-type TR2C. In contrast, the calcium-bound states are shown to undergo equilibrium exchange between at least two, approximately equally populated, conformations similar to those of the calcium-free and calcium- loaded states of wild-type TR2C. This conformational exchange occurs on the sub-millisecond time scale. From studies of the backbone fluctuations on pico- second to nanosecond time scales of the calcium- loaded state of the E140Q-mutant, the presence of any significantly populated unfolded intermediate substates could be excluded. Investigations of the temperature dependence of slower dynamics on micro- second to millisecond time scales indicated that the exchange does not involve one single structural transition. The use of off-resonance rotating frame spin relaxation experiments yielded an improved description of the exchange.