Refined structure of villin 14T and a detailed comparison with other actin-severing domains

Villin 14T is the amino terminal actin monomer binding domain from the actin-severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin-severing proteins. Protein Science 3:70-81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin-severing domain, severin domain 2.     

Probing the backbone dynamics of oxidized and reduced rat microsomal cytochrome b5 via 15N rotating frame NMR relaxation measurements: biological implications

Rotating frame 15N relaxation NMR experiments have been performed to study the local mobility of the oxidized and reduced forms of rat microsomal cytochrome b5, in the microsecond to millisecond time range. Measurements of rotating frame relaxation rates (R1rho) were performed as a function of the effective magnetic field amplitude by using off-resonance radio frequency irradiation. Detailed analysis of the two data sets resulted in the identification of slow motions along the backbone nitrogens for both oxidation states of the protein. The local mobility of reduced and oxidized cytochrome b5 turned out to be significantly different; 28 backbone nitrogens of the oxidized form were shown to participate in a conformational exchange process, while this number dropped to 12 in the reduced form. The correlation time, tauex, for the exchange processes could be determined for 21 and 9 backbone nitrogens for oxidized and reduced cytochrome b5, respectively, with their values ranging between 70 and 280 microseconds. The direct experimental evidence provided in this study for the larger mobility of the oxidized form of the protein is consistent with the different backbone NH solvent exchangeability recently documented for the two oxidation states [Arnesano, F., et al. (1998) Biochemistry 37, 173-184]. Our experimental observations may have significant biological implications. The differential local mobility between the two oxidation states is proposed to be an important factor controlling the molecular recognition processes in which cytochrome b5 is involved.