Backbone dynamics of the regulatory domain of calcium vector protein, studied by N-15 relaxation at four fields, reveals unique mobility characteristics of the intermotif linker

CaVP is a calcium-binding protein from amphioxus. It has a modular composit ion with two domains, but only the two EF-hand motifs localized in the C-te rminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca2+-saturated form and characteri zed the stepwise ion binding. This paper reports the N-15 nuclear relaxatio n rates of the Ca2+-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral densi ty function for the majority of the amide H-N-N vectors. Fitting the spectr al density values at eight frequencies by a model-free approach, we obtaine d the microdynamic parameters characterizing the global and internal moveme nts of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobilit y as reflected in the quite uniform order parameter and short internal corr elation time (< 20 nsec). Comparative analysis of the two Ca2+ binding site s shows that site III, having a larger affinity for the metal ion, is gener ally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneou sly by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP c haracterized here is discussed in relation with other well-characterized Ca 2+-binding proteins.