NMR Structural Studies of the C-Domain of Tcb2, A Calcium Binding Protein from Tetrahymena Thermophila

Abstract

Many protist cells use an ATP-independent form of contractile motility, which results from the calcium-triggered contraction of filaments containing novel structural and/or calcium-binding proteins. We have previously identified several such proteins during proteomic analysis of contractile fibers isolated from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila, including the putative calcium-binding protein Tcb2 and the filament-forming structural protein Epc1. To gain insight into the structure-function relationship of Tcb2, we have engineered genes for optimized bacterial expression of the full-length protein, as well as its isolated N- and C-terminal domains. Interestingly, a calcium-modulated contractile fabric can be reconstituted with purified full-length Tcb2. The full-length protein and its N-terminal domain are not amenable to high-resolution structure determination, as they tend to aggregate in the presence of calcium and/or upon concentration. However, the C-terminal domain (Tcb2-C) is highly soluble at both low and high calcium concentrations. Solution NMR HSQC spectra of 15N-labeled Tcb2-C indicate that the protein is well folded in the presence and absence of calcium and undergoes a dramatic conformational change upon calcium addition. We expressed and purified 15N, 13C-labeled Tcb2-C, and obtained nearly complete chemical shift assignments for the protein in both the presence and absence of calcium. The solution structure of calcium-free Tcb2-C was determined using NMR-derived experimental distance and torsion angle restraints. The current structural models reveal an architecture exhibited by other calcium-binding proteins, with paired EF-hand motifs connected by a flexible loop. NMR structure determination of calcium-bound Tcb2-C is currently underway. We are also using NMR spectroscopy to quantify the calcium-binding properties of the domain and investigate its conformational dynamics. These studies will establish a structural basis for elucidating the function and unique contractile properties of Tcb2.