Sequence-specific assignments of the backbone 1H, 13C, and 15N resonances of the MutT enzyme by heteronuclear multidimensional NMR.
The MutT protein, a 129-residue enzyme from Escherichia coli which prevents A.T-->C.G mutations, catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleoside monophosphates (NMP) and pyrophosphate [Bhatnagar, S. K., Bullions, L. C., & Bessman, M. J. (1991) J. Biol. Chem. 266, 9050-9054], by a mechanism involving nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. C., Bessman, M. J., & Mildvan, A. S. (1992a) J. Biol. Chem. 267, 16939-16942]. The bacterial MutT gene was inserted into the plasmid pET-11b under control of the T7 promoter and overexpressed in minimal media, permitting labeling of MutT with 13C and/or 15N. The yield after purification of the soluble fraction was approximately 35 mg of homogeneous MutT/L with physical and enzymatic properties indistinguishable from those of the originally isolated enzyme. Essentially complete sequence-specific assignments of the backbone HN, N, C alpha, H alpha, and CO resonances of the free enzyme (1.5 mM) were made at pH 7.4 and 32 degrees C, by heteronuclear double- and triple-resonance experiments using a modified Bruker AM 600 NMR spectrometer. Specifically, 1H[15N]HSQC, 1H[15N]TOCSY-HMQC, and 1H[15N]NOESY-HMQC experiments were done with uniformly 15N-labeled enzyme. A 1H[15N] HSQC experiment was done with selective [alpha-15N]Lys-labeled enzyme. Also HNCA, HN(CO)CA, HNCO, constant time 1H[13C]HSQC, HCACO, and HCA(CO)N experiments were done with uniformly 13C- and 15N-labeled enzyme. Sequence-specific assignments were initiated from HN and 15N chemical shifts of Gly residues and of selectively labeled Lys residues in 1H[15N]HSQC experiments. They were confirmed by C alpha chemical shifts of Ala residues uniquely identified by residual coupling to C beta resonances in constant time 1H[13C]HSQC experiments. The sequence-specific assignments proceeded bidirectionally, terminating at Pro residues and at residues with undetectable NH signals, and the segments were linked to complete the backbone assignments. The backbone assignments reported here have permitted the interpretation of NOEs in the elucidation of the solution secondary structure of MutT, and the C alpha and H alpha chemical shifts have provided an independent approach to identifying secondary structural elements and to define their extent [Weber, D. J., Abeygunawardana, C., Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (following paper in this issue)].
Abeygunawardana
C
C
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Weber
D J
DJ
Frick
D N
DN
Bessman
M J
MJ
Mildvan
A S
AS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases; D017433:Protein Structure, Secondary; D011755:Pyrophosphatases; D011994:Recombinant Proteins; D012997:Solvents
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012997:Solvents; D003903:Deuterium; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C052211:mutT protein, E coli