Masino, Laura; (2000) Spectroscopic studies of the conformational stability and ligand binding properties of calmodulin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this work the conformational stability of calmodulin (CaM) and its tryptic fragments has been investigated using optical spectroscopic techniques such as far-UV circular dichroism, fluorescence, and absorption spectroscopy. CaM is a ubiquitous eukaryotic calcium binding protein that consists of two structurally similar globular domains connected by a flexible linker, and each containing a pair of helix-loop-helix calcium binding motifs. The results of chemical and thermal denaturation experiments show that the stability of CaM and its isolated domains in the absence of calcium is relatively low and is strongly dependent on ionic strength and temperature. This raises the question of how apo-CaM is stabilised in vivo at resting calcium concentrations. In the presence of ligands such as calcium, magnesium, and target peptides, the stability of CaM is greatly enhanced, as predicted by the ligand binding theory. The extent of the stabilising effect depends on the free ligand concentration and on the affinity of the native and denatured states of CaM for the ligand. The interactions of CaM with magnesium have been investigated and the results show that magnesium competes with calcium for the EF- hand sites and reduces the affinity of Ca2+ -CaM for targets. As a consequence, magnesium amplifies the intrinsic differences in affinity of the N- and C-domains for calcium and for target sequences. Thus, CaM is extremely sensitive to general environmental conditions as well as to specific ligand interactions. Chemical denaturation studies also show that the behaviour of GuHCl is very complex, owing to its ionic strength contribution and to competition with Ca2+ -binding. Therefore urea may be more appropriate in the study of the stability of calmodulin and of proteins that bind metal ions or that are sensitive to changes in the ionic strength of the solution. Finally, there is experimental evidence that the stability of the isolated domains of CaM is significantly different from that of the domains in the intact protein. This suggests the presence of inter-domain interactions during the process of unfolding. This observation is confirmed by the analysis of the stability of the [beta]-sheet mutants of CaM, which shows that the structural and Ca2+-binding properties of the non-mutated domain can be affected by the mutation on the other domain.

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