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1 H, 15 N, and 13 C backbone chemical shift assignments, secondary structure, and magnesium‐binding characteristics of the bacillus subtilis response regulator, SpoOF, determined by heteronuclear high‐resolution NMR
Author(s) -
Feher Victoria A.,
Zapf James W.,
Hoch James A.,
Dahlquist Frederick W.,
Whiteley John M.,
Cavanagh John
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040915
Subject(s) - heteronuclear single quantum coherence spectroscopy , chemistry , crystallography , protein secondary structure , bacillus subtilis , stereochemistry , chemical shift , nuclear magnetic resonance spectroscopy , biology , biochemistry , bacteria , genetics
SpoOF, 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 1 H, 15 N, and 13 C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15 N TOCSY‐HSQC, 15 N 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 HNα coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound ( α/β ) 5 fold. Interestingly, comparison of the secondary structure of SpoOF to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266 :15511–15519; Bruix M et al., 1993, Eur J Biochem 2/ 5 :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 SpoOF, 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.