Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.
Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].
Fairbrother
W J
WJ
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Palmer
A G
AG
Rance
M
M
Reizer
J
J
Saier
M H
MH
Wright
P E
PE
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology*; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000201:enzymology; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D011487:Protein Conformation; D014961:X-Ray Diffraction
D002247:Carbon Isotopes; D006859:Hydrogen; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase