Polypeptide backbone resonance assignments and secondary structure of Bacillus subtilis enzyme III sup glc determined by two-dimensional and three-dimensional heteronuclear NMR spectroscopy
- Research Inst. of Scripps Clinic, La Jolla, CA (United States)
- Univ. of California, San Diego (United States)
The enzyme III{sup glc}-like domain of Bacillus subtilis II{sup glc} (III{sup glc}; 162 residues, 17.4 kDa) has been cloned and overexpressed in Escherichia coli. Sequence-specific assignment of the backbone {sup 1}H and {sup 15}N resonances has been carried out with a combination of mohonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional (3D) NMR spectroscopy. Amide proton solvent exchange rate constants have been determined from a series of {sup 1}H-{sup 15}N heteronuclear single-quantum coherence (HSQC) spectra acquired following dissolution of the protein in D{sub 2}O. Major structural features of III{sup glc} have been inferred from the pattern of short-, medium- and long-range NOEs in 3D heteronuclear {sup 1}H nuclear Overhauser effect {sup 1}H-{sup 15}N multiple-quantum coherence (3D NOESY-HMQC) spectra, together with the exchange rate constants. III{sup glc} contains three antiparallel {beta}-sheets comprised of eight, three, and two {beta}-strands. In addition, five {beta}-bulges were identified. no evidence of regular helical structure was found. The N-terminal 15 residues of the protein appear disordered, which is consistent with their being part of the Q-linker that connects the C-terminal enzyme III{sup glc}-like domain to the membrane-bound II{sup glc} domain. Significantly, two histidine residues, His 68 and His 83, which are important for phosphotransferase function, are found from NOE measurements to be in close proximity at the ends of adjacent strands in the major {beta}-sheet.
- OSTI ID:
- 5013835
- Journal Information:
- Biochemistry; (United States), Vol. 30:28; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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PHOSPHOTRANSFERASES
MOLECULAR STRUCTURE
POLYPEPTIDES
NUCLEAR MAGNETIC RESONANCE
BACILLUS SUBTILIS
HEAVY WATER
NITROGEN 15
OVERHAUSER EFFECT
PROTONS
BACILLUS
BACTERIA
BARYONS
ELEMENTARY PARTICLES
ENZYMES
FERMIONS
HADRONS
HYDROGEN COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
NITROGEN ISOTOPES
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PEPTIDES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
RESONANCE
STABLE ISOTOPES
TRANSFERASES
WATER
550601* - Medicine- Unsealed Radionuclides in Diagnostics