NMR-Spectroscopic Mapping of an Engineered Cavity in the I14A Mutant of HPr fromStaphylococcuscarnosusUsing Xenon

Gröger, Christian; Möglich, Andreas; Pons, Miquel; Koch, Brigitte; Hengstenberg, Wolfgang; Kalbitzer, Hans Robert; Brunner, Eike

doi:10.1021/ja030113t

Cited by 37 publications

(59 citation statements)

References 20 publications

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“…3). For most amino acid residues, the replacement of NH þ 4 by Na þ did not influence the 15 N chemical shifts as is demonstrated in Fig. 3 (top) for Cys9.…”

Section: Interaction Of Nh 3 With Hisf and Steady-state Enzyme Kineticsmentioning

confidence: 70%

“…The NMR signals of numerous amino acids are influenced by NH 4 Cl. In particular, pronounced 15 N chemical shift changes are observed (see Fig. 3), which can be described by a one site binding model (see Fig.…”

Section: Interaction Of Nh 3 With Hisf and Steady-state Enzyme Kineticsmentioning

confidence: 89%

“…On the basis of the existing spectral assignment, 1 H- 15 N HSQC experiments on the xenon-pressurized sample were used to identify and localize xenoninduced 1 H and/or 15 N chemical shift changes by comparison with the xenon-free sample. Such chemical shifts sensitively indicate minor local structural changes of the protein induced by specific binding of xenon.…”

Section: Identification Of Xenon Binding Sitesmentioning

confidence: 99%

“…Such chemical shifts sensitively indicate minor local structural changes of the protein induced by specific binding of xenon. [14][15][16][17][18] In our experiments, the sample was equilibrated at 11 bar xenon pressure. The spectral assignment was based on the data published by Lipchock and Loria 8 as well as our own work.…”

Section: Identification Of Xenon Binding Sitesmentioning

confidence: 99%

“…5,6 The active sites of HisH and HisF are separated by a distance of $25 Å . 6,7 Solution NMR spectroscopy was used to assign the 1 H, 15 N, and 13 C resonances of HisF and to study the influence of ImGP binding and complex formation between the two subunits on the protein dynamics of HisF. 8,9 It has been proposed that NH 3 is transported between the active sites of HisH and HisF through an ammonia channel, which includes the interior of the central barrel of HisF.…”

Section: Introductionmentioning

confidence: 99%

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The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy

et al. 2010

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The imidazole glycerol phosphate (ImGP) synthase from the hyperthermophilic bacterium Thermotoga maritima is a 1:1 complex of the glutaminase subunit HisH and the cyclase subunit HisF. It has been proposed that ammonia generated by HisH is transported through a channel to the active site of HisF, which generates intermediates of histidine (ImGP) and de novo biosynthesis of 5-aminoimidazole-4-carboxamideribotide. Solution NMR spectroscopy of ammonium chloride-titrated samples was used to study the interaction of NH 3 with amino acids inside this channel. Although numerous residues showed 15 N chemical shift changes, most of these changes were caused by nonspecific ionic strength effects. However, several interactions appeared to be specific. Remarkably, the amino acid residue Thr 78-which is located in the central channel-shows a large chemical shift change upon titration with ammonium chloride. This result and the reduced catalytic activity of the Thr78Met mutant indicate a special role of this residue in ammonia channeling. To detect and further characterize internal cavities in HisF, which might for example contribute to ammonia channeling, the interaction of HisF with the noble gas xenon was analyzed by solution NMR spectroscopy using 1 H-15 N HSQC experiments. The results indicate that HisF contains three distinct internal cavities, which could be identified by xenon-induced chemical shift changes of the neighboring amino acid residues. Two of these cavities are located at the active site at opposite ends of the substrate N 0 -[(5 0 -phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamideribonucleotide (PRFAR) binding groove. The third cavity is located in the interior of the central b-barrel of HisF and overlaps with the putative ammonia transport channel.

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“…3). For most amino acid residues, the replacement of NH þ 4 by Na þ did not influence the 15 N chemical shifts as is demonstrated in Fig. 3 (top) for Cys9.…”

Section: Interaction Of Nh 3 With Hisf and Steady-state Enzyme Kineticsmentioning

confidence: 70%

Section: Interaction Of Nh 3 With Hisf and Steady-state Enzyme Kineticsmentioning

confidence: 89%

Section: Identification Of Xenon Binding Sitesmentioning

confidence: 99%

Section: Identification Of Xenon Binding Sitesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy

et al. 2010

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Design of a Conformation‐Sensitive Xenon‐Binding Cavity in the Ribose‐Binding Protein

Lowery¹,

Rubin²,

Ruiz³

et al. 2004

Angewandte Chemie

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129Xe‐NMR‐Spektroskopie trifft Protein‐Engineering: NMR‐Spektroskopie mit Laser‐polarisiertem 129Xe hilft nur beim Nachweis von Proteinkonformationen, wenn das Protein über eine konformativ empfindliche Xenon bindende Kavität verfügt. Diese fehlt jedoch vielen Proteinen. Der vorgestellte Designprozess führte zu einer entsprechenden Kavität im Ribose bindenden Protein (siehe Bild) und damit zum 129Xe‐NMR‐spektroskopischen Nachweis von Proteinkonformation und Ligandenbindung.

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Design of a Conformation‐Sensitive Xenon‐Binding Cavity in the Ribose‐Binding Protein

et al. 2004

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129Xe NMR spectroscopy meets protein engineering: NMR spectroscopy with laser‐polarized 129Xe can be used to report protein conformations only if a protein has a conformationally sensitive xenon‐binding cavity. However, many proteins lack such reporter sites. A straightforward design process is used to engineer a xenon‐binding cavity into the ribose‐binding protein (see picture) that allows the 129Xe NMR spectroscopic report of protein conformation and ligand binding.

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NMR-Spectroscopic Mapping of an Engineered Cavity in the I14A Mutant of HPr fromStaphylococcuscarnosusUsing Xenon

Cited by 37 publications

References 20 publications

The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy

The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy

Design of a Conformation‐Sensitive Xenon‐Binding Cavity in the Ribose‐Binding Protein

Design of a Conformation‐Sensitive Xenon‐Binding Cavity in the Ribose‐Binding Protein

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