Related ArticlesInvolvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins.
The phosphocarrier HPr (heat stable protein) of Staphylococcus carnosus was modified by site-directed mutagenesis of the corresponding ptsH gene in order to analyse the importance of amino acids which were supposed to be part of the active centre of the protein. Three residues which are conserved in all HPrs, Arg17, Pro18 and Glu84, were mutated: Arg17 was changed to His (17RH) and Pro18 and Glu84 were changed into Ala (18PA and 84EA). In addition, Leu86 was changed into Ala (86LA) and one mutant protein was missing the last six residues of the HPr (delta 83). The wild type gene and all mutant genes were overexpressed and the gene products purified to homogeneity. Three-dimensional structures of wild type and mutant proteins were monitored by NMR spectroscopy. All five mutant HPrs had native conformations. The ATP-dependent HPr kinase can phosphorylate all HPr derivatives at Ser46. The PTS activity of the amino-terminal HPr mutant proteins 17RH and 18PA was different compared to wild type HPr. In contrast, the carboxy-terminal mutant HPrs possessed a similar enzyme activity to the wild type HPr. The 17RH and 18PA HPrs with substitution near the active centre His15 showed a very slow phosphorylation by enzyme I but the further transfer of the phosphoryl group to enzyme III was also strongly inhibited. The enzyme activity of the HPr 17RH was significantly improved at low pH. NMR pH-titration experiments showed that Arg17 is not responsible for the low pKa of the active centre His15 but this positively charged residue is essential in this position for the HPr activity.
Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study.
Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study.
Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study.
Biochemistry. 2011 Aug 27;
Authors: Peng D, Satterlee JD, Ma LH, Dallas JL, Smith KM, Zhang X, Sato M, La Mar GN
Abstract
Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which...
Site-specific labeling of proteins with NMR-active unnatural amino acids
Site-specific labeling of proteins with NMR-active unnatural amino acids
Abstract A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling...
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[NMR paper] The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic
The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.
Related Articles The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis.
Biochemistry. 2004 Apr 13;43(14):4082-91
Authors: Azurmendi HF, Wang SC, Massiah MA, Poelarends GJ, Whitman CP, Mildvan AS
trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the...
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11-24-2010 09:51 PM
[NMR paper] Measurement of side-chain carboxyl pK(a) values of glutamate and aspartate residues i
Measurement of side-chain carboxyl pK(a) values of glutamate and aspartate residues in an unfolded protein by multinuclear NMR spectroscopy.
Related Articles Measurement of side-chain carboxyl pK(a) values of glutamate and aspartate residues in an unfolded protein by multinuclear NMR spectroscopy.
J Am Chem Soc. 2002 May 22;124(20):5714-7
Authors: Tollinger M, Forman-Kay JD, Kay LE
A triple-resonance NMR pulse scheme is presented for measuring aspartic and glutamic acid side-chain pK(a) values in unfolded protein states where chemical shift...
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[NMR paper] Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and ir
Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
Related Articles Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
J Biomol NMR. 2000 Aug;17(4):311-22
Authors: Sorkin DL, Miller AF
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide...
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[NMR paper] Modulation of intrinsic phi,psi propensities of amino acids by neighbouring residues
Modulation of intrinsic phi,psi propensities of amino acids by neighbouring residues in the coil regions of protein structures: NMR analysis and dissection of a beta-hairpin peptide.
Related Articles Modulation of intrinsic phi,psi propensities of amino acids by neighbouring residues in the coil regions of protein structures: NMR analysis and dissection of a beta-hairpin peptide.
J Mol Biol. 1998 Dec 18;284(5):1597-609
Authors: Griffiths-Jones SR, Sharman GJ, Maynard AJ, Searle MS
Analysis of residues in coil regions of protein structures...
Involvement of various amino- and carboxyl-terminal residues in the active site of th
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