Near‐Atomic Resolution Neutron Crystallography on Perdeuterated <i>Pyrococcus furiosus</i> Rubredoxin: Implication of Hydronium Ions and Protonation State Equilibria in Redox Changes

Cuypers, M. G.; Mason, Sax A.; Blakeley, Matthew P.; Mitchell, Edward P.; Haertlein, Michael; Forsyth, V. T.

doi:10.1002/ange.201207071

Cited by 14 publications

(15 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the current study, perdeuterated Pyrococcus furiosus rubredoxin 18 was produced with the iron atom from the [Fe-4S] cluster replaced by the 113 Cd isotope. Neutron diffraction data were collected from a perdeuterated crystal ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Macromolecular structure phasing by neutron anomalous diffraction

Cuypers

Mason²,

Mossou

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

In this report we show for the first time that neutron anomalous dispersion can be used in a practical manner to determine experimental phases of a protein crystal structure, providing a new tool for structural biologists. The approach is demonstrated through the use of a state-of-the-art monochromatic neutron diffractometer at the Institut Laue-Langevin (ILL) in combination with crystals of perdeuterated protein that minimise the level of hydrogen incoherent scattering and enhance the visibility of the anomalous signal. The protein used was rubredoxin in which cadmium replaced the iron at the iron-sulphur site. While this study was carried out using a steady-state neutron beam source, the results will be of major interest for capabilities at existing and emerging spallation neutron sources where time-of-flight instruments provide inherent energy discrimination. In particular this capability may be expected to offer unique opportunities to a rapidly developing structural biology community where there is increasing interest in the identification of protonation states, protein/water interactions and protein-ligand interactions – all of which are of central importance to a wide range of fundamental and applied areas in the biosciences.

show abstract

Section: Resultsmentioning

confidence: 99%

Macromolecular structure phasing by neutron anomalous diffraction

Cuypers

Mason²,

Mossou

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

show abstract

“…Simultaneously, the six-fold decrease in the D incoherent scattering length (3.99 fm) compared to H (25.27 fm) dramatically reduces the isotropic background intensity. The gain in signal and reduction in noise allow datasets to be collected from smaller crystals (< 0.5 mm 3 ) or with reduced total exposure times (< 10 days) [13,20,[36][37][38]. Furthermore, the D coherent scattering length is positive as is that of common atoms found in proteins while the coherent scattering length of H is negative (Figure 1).…”

Section: Hydrogen/deuterium Isotopic Substitutionmentioning

confidence: 99%

“…In order to exchange the remaining 75% attached to carbon atoms, proteins need to be perdeuterated during synthesis. Expression of perdeuterated protein in fully D-labeled growth media has yielded neutron protein crystal structures currently deposited in the PDB for six proteins, namely myoglobin [64,65], aldose reductase [13,41], transthyretin [38], type III antifreeze protein [66,67], beta lactamase [19,20] and rubredoxin [3,36,37,68,69].…”

Section: Perdeuterationmentioning

confidence: 99%

“…[36] Table 2 lists current single crystal diffractometers designed for neutron protein crystallography at neutron scattering facilities worldwide. In addition to these instruments, other multi-purpose single crystal diffractometers, such as the D19 instrument at ILL, have also been used for protein crystallography [5,28,37,38]. Instruments can be divided into three groups based upon mode of operation, namely monochromatic, quasi-Laue (also known as "pink-Laue") and time-of-flight (TOF) Laue modes.…”

Section: Instrumentation Data Collection and Processingmentioning

confidence: 99%

“…An initial rigid body refinement and successive coordinate, atomic displacement parameter and occupancy refinements are performed in alternation with local rebuilding of the model including the addition of small molecules. Water molecules can be modeled as O, OD, OD -, DOD, and DO 3 + depending on their degree of rotational anisotropy, neighboring groups, and ionic state [32,37,150]. Water molecules can be oriented automatically by the refinement software.…”

Section: Structure Refinementmentioning

confidence: 99%

See 2 more Smart Citations

Neutron protein crystallography: A complementary tool for locating hydrogens in proteins

O’Dell

Bodenheimer

Meilleur

2016

Archives of Biochemistry and Biophysics

103

View full text Add to dashboard Cite

Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.

show abstract

Long‐Range Electrostatics‐Induced Two‐Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site

et al. 2016

View full text Add to dashboard Cite

Proton transfer is integral to many biochemical processes. However, the direct observation and structural characterization of biological proton transfer has hitherto not been possible. Here, neutron crystallography directly locates two protons before and after a pH-induced two-proton transfer between the catalytic aspartic acid residues and the hydroxyl group of the bound clinical drug, darunavir, in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by QM/MM calculations. We propose that the low-pH proton configuration in the catalytic site is critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.

show abstract

Near‐Atomic Resolution Neutron Crystallography on Perdeuterated Pyrococcus furiosus Rubredoxin: Implication of Hydronium Ions and Protonation State Equilibria in Redox Changes

Cited by 14 publications

References 19 publications

Macromolecular structure phasing by neutron anomalous diffraction

Macromolecular structure phasing by neutron anomalous diffraction

Neutron protein crystallography: A complementary tool for locating hydrogens in proteins

Long‐Range Electrostatics‐Induced Two‐Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site

Contact Info

Product

Resources

About