Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.
kinase (AK) gaining access to varying populations of the LU state. With nuclear magnetic resonance experiments we are able to observe structural changes in the LID upon mutation consistent with local unfolding. More importantly conformational dynamics were measured from the both the folded and locally unfolded states giving access to the populations of both states and the kinetics of the folding/unfolding transition. Furthermore, the changes in population upon mutation are driven by an increase in the unfolding rate of the LID while enzyme kinetics are unaffected at physiological temperatures, evidence that LID unfolding is not rate limiting in the catalytic reaction. These experiments allow insight into the protein dynamics that drive function and exemplify the ensemble nature of the native state and the importance of conformational fluctuations in tuning biological activity.
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