Neutron crystallography of rubredoxin

Kurihara, Kazuo; Tanaka, I.; Adams, M.W.W.; Moiseeva, Natalia; Bau, Robert; Niimura, S.

doi:10.2142/biophys.40.s167_4

Cited by 6 publications

(15 citation statements)

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“…Large crystals of mut-PfRd, partially deuterated in this manner, were grown under the same conditions as those used for wild-type PfRd: i.e. using 3.6 M sodium/potassium phosphate (in D 2 O) as a precipitant with seeding (Bau et al, 1998;Kurihara et al, 2001).…”

Section: Sample Preparation and Crystallizationmentioning

confidence: 99%

“…In the present study, we succeeded in determining almost all of the hydrogen and deuterium positions in mut-PfRd. Previously, we had solved the neutron crystal structure of wildtype PfRd at 1.5 A Ê resolution, also using the BIX-3 diffractometer (Kurihara et al, 2001(Kurihara et al, , 2004. In this paper, we describe three sets of results: (i) we show a comparison between the two neutron structures (PfRd and mut-PfRd), (ii) we map out the H/D distributions of the backbone NÐH bonds obtained after partial H/D exchange by exposing the protein solution to D 2 O and (iii) we describe the detailed appearance of water molecules of hydration near the surface of the protein.…”

Section: Introductionmentioning

confidence: 99%

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A neutron crystallographic analysis of a rubredoxin mutant at 1.6 Å resolution

Chatake

Kurihara

Tanaka

et al. 2004

Acta Crystallogr D Biol Crystallogr

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A neutron diffraction study has been carried out at 1.6 A resolution on a mutant rubredoxin from Pyrococcus furiosus using the BIX-3 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Research Institute. In order to study the unusual thermostability of rubredoxin from P. furiosus (an organism that grows optimally at 373 K), the hydrogen-bonding patterns were compared between the wild-type protein and a 'triple-mutant' variant. In this mutant protein, three residues were changed (Trp3-->Tyr3, Ile23-->Val23, Leu32-->Ile32) so that they are identical to those in a mesophilic rubredoxin from Clostridium pasteurianum. In the present study, some minor changes were found between the wild-type and mutant proteins in the hydrogen-bonding patterns of the Trp3/Tyr3 region. In this investigation, the H/D-exchange ratios in the protein were also studied. Because the target protein was soaked in D2O during the crystallization procedure, most of the N-H and O-H bonds have become deuterated, while essentially all of the C-H bonds have not. In particular, the H/D-exchange pattern of the N-H amide bonds of the protein backbone is of interest because it may contain some indirect information about the mechanism of unfolding of this small protein. The results are in broad agreement with those from solution NMR studies, which suggest that the backbone amide bonds near the four Cys residues of the FeS4 redox center are most resistant to H/D exchange. Finally, the detailed geometries of the water molecules of hydration around the rubredoxin molecule are also reported. The 1.6 A resolution of the present neutron structure determination has revealed a more detailed picture than previously available of some portions of the water structure, including ordered and disordered O-D bonds. Crystallographic details: space group P2(1)2(1)2(1) (orthorhombic), unit-cell parameters a = 34.48, b = 35.70, c = 43.16 A; final agreement factors R = 0.196 and Rfree = 0.230 for 19,384 observed and 6548 unique neutron reflections collected at room temperature; crystal size 4 mm3; a total of 423 non-H atoms, 290 H atoms and 88 D atoms were located in this study.

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Section: Sample Preparation and Crystallizationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A neutron crystallographic analysis of a rubredoxin mutant at 1.6 Å resolution

Chatake

Kurihara

Tanaka

et al. 2004

Acta Crystallogr D Biol Crystallogr

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“…The present study is the latest in a series designed to determine the contribution of hydrogen bonding, hydration and dynamics to the thermostability of PfRd (Kurihara et al, 2001;Chatake et al, 2002). It has been dif®cult to directly determine the positions of H atoms in the X-ray structures of rubredoxin and none have been reported for the labile positions in the ultrahigh-resolution structure of PfRd (Bau et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…The neutron structures of the wild-type PfRd and a triple mutant have already been determined using data collected on BIX-3 at JAERI (Kurihara et al, 2001;Chatake et al, 2002). This preliminary report concerns the neutron structure of the W3Y (Trp33Tyr3) single-mutant PfRd determined from data collected at the spallation neutron Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center.…”

Section: Introductionmentioning

confidence: 99%

W3Y single mutant of rubredoxin fromPyrococcus furiosus: a preliminary time-of-flight neutron study

Langan

Bau

et al. 2003

Acta Crystallogr D Biol Cryst

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Rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus maintains its native structure at high temperatures (373 K). In order to investigate the role of hydrogen bonding, hydration and chain dynamics in this thermostability, wavelength-resolved Laue neutron diffraction data have been collected from the W3Y single mutant (Trp33Tyr3) on the spallation neutron protein crystallography station (PCS) at Los Alamos Neutron Science Center. Data were measured at room temperature from nine crystal settings, each of approximately 12 h duration. The total data-measurement period was less than 5 d from a single crystal that had undergone H 2 O/D 2 O exchange. The nominal resolution of the data is 2.1 A Ê .

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“…All of these features have been incorporated in a neutron-imaging-plate-based diffractometer called BIX-3 (19)(20)(21) that has been built and installed at the JRR-3M reactor of the Japan Atomic Energy Research Institute. The present study on rubredoxin was performed by using this diffractometer (22,23), and subsequent studies with sperm whale myoglobin (to a 1.5-Å resolution) (22,24,25) and a rubredoxin mutant (to a 1.6-Å resolution) (26,27) have followed. A preliminary account of the work described in this article appeared in ref.…”

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confidence: 99%