A neutron crystallographic analysis of a rubredoxin mutant

In order to study the unusual thermostability of rubredoxin from Pyrococcus furiosus (RubPf), the structure of a \u27triple mutant\u27 of this rubredoxin, which is less thermostable than the wild type, was solved at 1.5-Å resolution by neutron-diffraction analysis using the BIX-3 diffractometer at the JRR-3M reactor of JAERI. The positions of the non-hydrogen atoms are almost the same as the native rubredoxin; however, for Trp3 → Tyr3, large structural changes were found, so that their hydrogen-bonding schemes are significantly different. Some positions of the hydrogen atoms and molecules of hydration are shifted in certain regions, suggesting that such differences may contribute to the differences in thermostability between this \u27triple-mutant\u27 rubredoxin and wild-type rubredoxin

A neutron crystallographic analysis of a rubredoxin mutant at 1.6 Ã… resolution

A neutron diffraction study has been carried out at 1.6 Å 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 \u27triple-mutant\u27 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 Å 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 P21212 1 (orthorhombic), unit-cell parameters a = 34.48, b = 35.70, c = 43.16 Å; 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. © 2004 International Union of Crystallography

Preliminary time-of-flight neutron diffraction study of human deoxyhemoglobin

In order to investigate the role of the protonation states of protein residues in O2 binding, large crystals of deoxy HbA (∼20 mm3) were grown in D2O under anaerobic conditions for neutron diffraction studies

Neutron crystallographic study on rubredoxin from Pyrococcus furiosus by BIX-3, a single-crystal diffractometer for biomacromolecules

The structure of a partially deuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus, an organism that grows optimally at 100°C, was determined by using the neutron single-crystal diffractometer dedicated for biological macromolecules (BIX-3) at the JRR-3M reactor of the Japan Atomic Energy Research Institute. Data were collected at room temperature up to a resolution of 1.5 Å, and the completeness factor of the data set was 81.9%. The model contains 306 H and 50 D atoms. A total of 37 hydration water molecules were identified, with 15 having all three atoms fully located and the remaining D(2)O molecules partially defined. The model has been refined to final agreement factors of R = 18.6% and R(free) = 21.7%. Several orientations of the O–D bonds of side chains, whose assignments from x-ray data were previously ambiguous, were clearly visible in the neutron structure. Although most backbone N–H bonds had undergone some degree of H/D exchange throughout the rubredoxin molecule, 5 H atom positions still had distinctly negative (H) peaks. The neutron Fourier maps clearly showed the details of an extensive set of H bonds involving the ND(3)(+) terminus that may contribute to the unusual thermostability of this molecule

Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography

HIV-1 protease is a dimeric aspartic protease that plays an essential role in viral replication. To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp-25 and Asp-125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9-Å resolution and X-ray crystallography at 1.4-Å resolution. The resulting structural data show that the catalytic residue Asp-25 is protonated and that Asp-125 (the catalytic residue from the corresponding diad-related molecule) is deprotonated. The proton on Asp-25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine (Apns) group in KNI-272. The deprotonated Asp-125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease and can therefore contribute substantially to the development of specific inhibitors for therapeutic application