H-1, N-15, AND C-13 BACKBONE CHEMICAL-SHIFT ASSIGNMENTS, SECONDARY STRUCTURE, AND MAGNESIUM-BINDING CHARACTERISTICS OF THE BACILLUS-SUBTILIS RESPONSE REGULATOR, SPOOF, DETERMINED BY HETERONUCLEAR HIGH-RESOLUTION NMR
Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The H-1, N-15, and C-13 chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, N-15 TOCSY-HSQC, N-15 NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many side-chain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, (3)J(HN alpha) coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)(5) fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volt K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, fur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.
Feher, V. A., Zapf, J. W., Hoch, J. A., DAHLQUIST, F. W., WHITELEY, J. M., & CAVANAGH, J. (1995). H-1, N-15, AND C-13 BACKBONE CHEMICAL-SHIFT ASSIGNMENTS, SECONDARY STRUCTURE, AND MAGNESIUM-BINDING CHARACTERISTICS OF THE BACILLUS-SUBTILIS RESPONSE REGULATOR, SPOOF, DETERMINED BY HETERONUCLEAR HIGH-RESOLUTION NMR. Protein Science, 4(9), 1801-1814.