Neutron Laue diffraction study of concanavalin A The proton of Asp28

Habash, J.; Raftery, James; Weisgerber, S.; Cassetta, Alberto; Lehmann, M. S.; Hghj, Peter; Wilkinson, C.; Campbell, John W.; Helliwell, John R.

doi:10.1039/a704143h

Cited by 48 publications

(56 citation statements)

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“…Indeed, it was this high resolution of the data which made it possible (Deacon et al, 1997) to distinguish in the unrestrained re®nement between bond lengths 1.170 (9) A Ê and 1.324 (10) A Ê as C O and CÐO(H) in a key carboxyl group, Asp28. The result was con®rmed by the detection of the H atom both in the X-ray and in a neutron Laue analysis (Habash et al, 1997).…”

Section: More About Restraintsmentioning

confidence: 84%

Remarks about protein structure precision

Cruickshank

1999

Acta Crystallogr D Biol Crystallogr

513

482

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Full‐matrix least squares is taken as the basis for an examination of protein structure precision. A two‐atom protein model is used to compare the precisions of unrestrained and restrained refinements. In this model, restrained refinement determines a bond length which is the weighted mean of the unrestrained diffraction‐only length and the geometric dictionary length. Data of 0.94 Å resolution for the 237‐residue protein concanavalin A are used in unrestrained and restrained full‐matrix inversions to provide standard uncertainties σ(r) for positions and σ(l) for bond lengths. σ(r) is as small as 0.01 Å for atoms with low Debye B values but increases strongly with B. The results emphasize the distinction between unrestrained and restrained refinements and between σ(r) and σ(l). Other full‐matrix inversions are reported. Such inversions require massive calculations. Several approximate methods are examined and compared critically. These include a Fourier map formula [Cruickshank (1949). Acta Cryst.2, 65–82], Luzzati plots [Luzzati (1952). Acta Cryst.5, 802–810] and a new diffraction‐component precision index (DPI). The DPI estimate of σ(r, Bavg) is given by a simple formula. It uses R or Rfree and is based on a very rough approximation to the least‐squares method. Many examples show its usefulness as a precision comparator for high‐ and low‐resolution structures. The effect of restraints as resolution varies is examined. More regular use of full‐matrix inversion is urged to establish positional precision and hence the precision of non‐dictionary distances in both high‐ and low‐resolution structures. Failing this, parameter blocks for representative residues and their neighbours should be inverted to gain a general idea of σ(r) as a function of B. The whole discussion is subject to some caveats about the effects of disordered regions in the crystal.

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Section: More About Restraintsmentioning

confidence: 84%

Remarks about protein structure precision

Cruickshank

1999

Acta Crystallogr D Biol Crystallogr

513

482

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“…The combination of a broad band-pass quasi-Laue geometry (e.g., λ ) 3.5 Å, dλ/λ ) 25%) with a novel cylindrical, neutron sensitive image plate detector that completely surrounds the sample provides 10-100-fold gains in efficiency compared with those of conventional monochromatic neutron diffractometers (21). This has made feasible studies of larger biological complexes and smaller crystals than was previously possible and has enabled the location of important hydrogen and/or deuterium atom positions in a number of systems at the resolutions typical of the majority of protein structure analyses (22)(23)(24)(25). This approach therefore offers great promise in the study and location of key hydrogen atom positions involved in enzymatic mechanisms, proton pumping, and shuttling processes and in protein-ligand bonding interactions.…”

Section: Background To Neutron Studiesmentioning

confidence: 99%

A Neutron Laue Diffraction Study of Endothiapepsin: Implications for the Aspartic Proteinase Mechanism

et al. 2001

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Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 Å to an R-factor of 23.5% and an R free of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D 2

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“…method (Habash et al 1997). This improves the signalto-noise ratio of the data and hence the resolution limit by significantly reducing the hydrogen incoherent scattering contribution to the background.…”

Section: Deuterationmentioning

confidence: 99%

Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states inAspergillus flavusurate oxidase

Oksanen

Blakeley

Bonneté

et al. 2009

J. R. Soc. Interface.

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Urate oxidase (Uox) catalyses the oxidation of urate to allantoin and is used to reduce toxic urate accumulation during chemotherapy. X-ray structures of Uox with various inhibitors have been determined and yet the detailed catalytic mechanism remains unclear. Neutron crystallography can provide complementary information to that from X-ray studies and allows direct determination of the protonation states of the active-site residues and substrate analogues, provided that large, well-ordered deuterated crystals can be grown. Here, we describe a method and apparatus used to grow large crystals of Uox (Aspergillus flavus) with its substrate analogues 8-azaxanthine and 9-methyl urate, and with the natural substrate urate, in the presence and absence of cyanide. High-resolution X-ray (1.05 -1.20 Å ) and neutron diffraction data (1.9 -2.5 Å ) have been collected for the Uox complexes at the European Synchrotron Radiation Facility and the Institut Laue-Langevin, respectively. In addition, room temperature X-ray data were also collected in preparation for joint X-ray and neutron refinement. Preliminary results indicate no major structural differences between crystals grown in H 2 O and D 2 O even though the crystallization process is affected. Moreover, initial nuclear scattering density maps reveal the proton positions clearly, eventually providing important information towards unravelling the mechanism of catalysis.Keywords: urate oxidase; neutron and X-ray crystallography; crystal growth; phase diagram; H -D exchange; protonation states INTRODUCTIONUrate oxidase (uricase or Uox, EC 1.7.3.3) is an enzyme involved in purine metabolism. It catalyses the oxidation of uric acid to 5-hydroxyisourate, a metastable intermediate, which is further degraded to allantoin. Humans and other primates lack a functional Uox, and therefore the final product of purine metabolism is uric acid. It has been speculated that this confers an evolutionary advantage to long-lived animals like primates owing to the anti-oxidant properties of urate, but the downside is that in certain conditions high urate levels (hyperuricaemia) may lead to crystallization of uric acid, as in gout.Uox from Aspergillus flavus has been commercialized by Sanofi-Aventis as a protein drug to treat hyperuraemic conditions such as the tumour lysis syndrome, but the catalytic mechanism of Uox is still poorly understood. Several X-ray structures have been solved for Uox in the complex with various uric acid analogues (Colloc'h et al. 1997(Colloc'h et al. , 2007Retailleau et al. 2004Retailleau et al. , 2005Gabison et al. 2006Gabison et al. , 2008, and a putative mechanism for the oxidation of uric acid has been proposed. However, many of the key questions are related to the protonation state of the substrate during the reaction. Uric acid has four potentially acidic protons; this gives rise to four different monoanions and six dianions. To be able to describe the mechanism, it is necessary to know which of the anions is the real substrate and how the hydrogen bonding netw...

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Neutron Laue diffraction study of concanavalin A The proton of Asp28

Cited by 48 publications

References 1 publication

Remarks about protein structure precision

Remarks about protein structure precision

A Neutron Laue Diffraction Study of Endothiapepsin: Implications for the Aspartic Proteinase Mechanism

Large crystal growth by thermal control allows combined X-ray and neutron crystallographic studies to elucidate the protonation states inAspergillus flavusurate oxidase

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