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1.
FEBS J ; 286(6): 1204-1213, 2019 03.
Article in English | MEDLINE | ID: mdl-30657257

ABSTRACT

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site-directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure-induced reactivation was due to the decrease in the volume of the system, -ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme.


Subject(s)
Aspartic Acid/metabolism , Bacteria/enzymology , Dihydroorotase/chemistry , Dihydroorotase/metabolism , Zinc/metabolism , Aquifex , Aspartic Acid/chemistry , Aspartic Acid/genetics , Catalytic Domain , Crystallography , Dihydroorotase/genetics , Mutagenesis, Site-Directed , Mutation , Pressure , Protein Conformation
2.
Curr Opin Struct Biol ; 22(5): 636-42, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22959123

ABSTRACT

Biomacromolecules are thermodynamic entities that exist in general as an equilibrium mixture of the basic folded state and various higher-energy substates including all functionally relevant ones. Under physiological conditions, however, the higher-energy substates are usually undetectable on spectroscopy, as their equilibrium populations are extremely low. Hydrostatic pressure gives a general solution to this problem. As proteins generally have smaller partial molar volumes in higher-energy states than in the basic folded state, pressure can shift the equilibrium toward the former substantially, and allows their direct detection and analysis with X-ray crystallography or NMR spectroscopy at elevated pressures. These techniques are now mature, and their status and selected applications are presented with future prospects.


Subject(s)
Crystallography, X-Ray/methods , Macromolecular Substances/chemistry , Magnetic Resonance Spectroscopy/methods , Pressure
3.
J Synchrotron Radiat ; 19(Pt 3): 323-31, 2012 May.
Article in English | MEDLINE | ID: mdl-22514165

ABSTRACT

The XPAD3S-CdTe, a CdTe photon-counting pixel array detector, has been used to measure the energy and the intensity of the white-beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy-resolved X-ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending-magnet beamline METROLOGIE at Synchrotron SOLEIL. The X-ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging-plate detector and then measured with both the XPAD3S-CdTe and the XPAD3S-Si, a silicon photon-counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit-cell parameter determination and the measurement of high-quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X-ray diffraction opened up by this type of technology are discussed.


Subject(s)
Crystallography, X-Ray/methods , Muramidase/chemistry , Animals , Calibration , Chickens , Photons , Synchrotrons/instrumentation
4.
J Synchrotron Radiat ; 18(1): 1, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21169679

ABSTRACT

An introductory overview to the special issue papers on diffraction structural biology in this issue of the journal.


Subject(s)
Molecular Biology , X-Ray Diffraction , Biology
5.
J Synchrotron Radiat ; 18(1): 31-6, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21169687

ABSTRACT

Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined.


Subject(s)
Crystallography, X-Ray/methods , Macromolecular Substances/chemistry , Synchrotrons/instrumentation , Muramidase/chemistry , Pressure , X-Rays
6.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 6): 654-63, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20516618

ABSTRACT

The 2 A resolution crystal structure of bovine erythrocyte Cu,Zn superoxide dismutase (CuZnSOD) has been determined by X-ray diffraction at high pressure (0.57 GPa) and room temperature. At 0.57 GPa the secondary, tertiary and quaternary structures are similar to other previously determined bovine erythrocyte CuZnSOD structures. Nevertheless, pressure has a localized impact on the atomic coordinates of C(alpha) atoms and on side chains. The compression of the crystal and of the protein backbone is anisotropic. This anisotropy is discussed, taking into account intermolecular contacts and protein conformation. Pressure perturbation highlights the more flexible zones in the protein such as the electrostatic loop. At 0.57 GPa, a global shift of the dimetallic sites in both subunits and changes in the oxidation state of Cu were observed. The flexibility of the electrostatic loop may be useful for the interaction of different metal carriers in the copper-uptake process, whereas the flexibility of the metal sites involved in the activity of the protein could contribute to explaining the ubiquitous character of CuZnSODs, which are found in organisms living in very different conditions, including the deep-sea environment. This work illustrates the potential of combining X-ray crystallography with high pressure to promote and stabilize higher energy conformational substates.


Subject(s)
Superoxide Dismutase/chemistry , Animals , Anisotropy , Cattle , Crystallography, X-Ray , Ligands , Models, Molecular , Pliability , Protein Structure, Quaternary , Protein Structure, Tertiary , Structural Homology, Protein
7.
Biophys J ; 98(10): 2365-73, 2010 May 19.
Article in English | MEDLINE | ID: mdl-20483346

ABSTRACT

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.


Subject(s)
Hydrostatic Pressure/adverse effects , Protein Conformation/radiation effects , Protein Denaturation/radiation effects , Structure-Activity Relationship , Urate Oxidase/radiation effects , Catalysis , Kinetics , Models, Molecular , Spectrometry, Fluorescence , Urate Oxidase/chemistry , Urate Oxidase/metabolism
8.
Annu Rev Biophys ; 38: 153-71, 2009.
Article in English | MEDLINE | ID: mdl-19416064

ABSTRACT

A survey of the main interests of high pressure for molecular biophysics highlights the possibility of exploring the whole conformational space using pressure perturbation. A better understanding of fundamental mechanisms responsible for the effects of high pressure on biomolecules requires high-resolution molecular information. Thanks to recent instrumental and methodological progress taking advantage of the remarkable adaptation of the crystalline state to hydrostatic compression, pressure-perturbed macromolecular crystallography is now a full-fledged technique applicable to a variety of systems, including large assemblies. This versatility is illustrated by selected applications, including DNA fragments, a tetrameric protein, and a viral capsid. Binding of compressed noble gases to proteins is commonly used to solve the phase problem, but standard macromolecular crystallography would also benefit from the transfer of experimental procedures developed for high-pressure studies. Dedicated short-wavelength synchrotron radiation beamlines are unarguably required to fully exploit the various facets of high-pressure macromolecular crystallography.


Subject(s)
Biophysics/trends , Biopolymers/chemistry , Crystallography/trends , Macromolecular Substances/chemistry , Specimen Handling/trends , Molecular Conformation , Pressure
9.
Nucleic Acids Res ; 35(14): 4800-8, 2007.
Article in English | MEDLINE | ID: mdl-17617642

ABSTRACT

The behaviour of the d(GGTATACC) oligonucleotide has been investigated by X-ray crystallography at 295 K in the range from ambient pressure to 2 GPa (approximately 20,000 atm). Four 3D-structures of the A-DNA form (at ambient pressure, 0.55, 1.09 and 1.39 GPa) were refined at 1.60 or 1.65 A resolution. In addition to the diffraction pattern of the A-form, the broad meridional streaks previously explained by occluded B-DNA octamers within the channels of the crystalline A-form matrix were observed up to at least 2 GPa. This work highlights an important property of nucleic acids, their capability to withstand very high pressures, while keeping in such conditions a nearly invariant geometry of base pairs that store and carry genetic information. The double-helix base-paired architecture behaves as a molecular spring, which makes it especially adapted to very harsh conditions. These features may have contributed to the emergence of a RNA World at prebiotic stage.


Subject(s)
DNA, A-Form/chemistry , DNA/chemistry , Models, Molecular , Oligodeoxyribonucleotides/chemistry , Base Pairing , Crystallography, X-Ray , Hydrostatic Pressure , Nucleic Acid Conformation
10.
Biochim Biophys Acta ; 1764(3): 384-90, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16487756

ABSTRACT

Recent technical developments, achievements and prospects of high-pressure (HP) macromolecular crystallography (MX) are reviewed. Technical difficulties associated with this technique have been essentially solved by combining synchrotron radiation of ultra-short wavelength, large-aperture diamond anvil cells and new sample-mounting techniques. The quality of diffraction data collected at HP can now meet standards of conventional MX. The exploitation of the potential of the combination of X-ray diffraction and high-pressure perturbation is progressing well. The ability of pressure to shift the population distribution of conformers in solution, which is exploited in particular by NMR, can also be used in the crystalline state with specific advantages. HPMX has indeed bright prospects, in particular to elucidate the structure of higher-energy conformers that are often of high biological significance. Furthermore, HPMX may be of interest for conventional crystallographic studies, as pressure is a fairly general tool to improve order in pre-existing crystals with minimal perturbation of the native structure.


Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Animals , Humans , Pressure , Protein Conformation , X-Ray Diffraction
11.
Biochim Biophys Acta ; 1764(3): 391-7, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16478683

ABSTRACT

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 A resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.


Subject(s)
Aspergillus flavus/enzymology , Enzyme Inhibitors/chemistry , Fungal Proteins/chemistry , Urate Oxidase/chemistry , Xanthines/chemistry , Atmospheric Pressure , Crystallography, X-Ray , Hydrostatic Pressure , Protein Conformation , Temperature , Urate Oxidase/antagonists & inhibitors , Water/chemistry
12.
Biophys J ; 88(5): 3562-71, 2005 May.
Article in English | MEDLINE | ID: mdl-15731378

ABSTRACT

The structure of cubic Cowpea mosaic virus crystals, compressed at 330 MPa in a diamond anvil cell, was refined at 2.8 A from data collected using ultrashort-wavelength (0.331 A) synchrotron radiation. With respect to the structure at atmospheric pressure, order is increased with lower Debye Waller factors and a larger number of ordered water molecules. Hydrogen-bond lengths are on average shorter and the cavity volume is strongly reduced. A tentative mechanistic explanation is given for the coexistence of disordered and ordered cubic crystals in crystallization drops and for the disorder-order transition observed in disordered crystals submitted to high pressure. Based on such explanation, it can be concluded that pressure would in general improve, albeit to a variable extent, the order in macromolecular crystals.


Subject(s)
Biophysics/methods , Capsid/chemistry , Comovirus/chemistry , Comovirus/metabolism , Crystallography, X-Ray , Hydrogen Bonding , Macromolecular Substances , Models, Molecular , Molecular Conformation , Pressure , Proteins/chemistry , Software , Surface Properties , Synchrotrons , Temperature , Water
13.
J Synchrotron Radiat ; 12(Pt 1): 1-3, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15616356

ABSTRACT

An overview of the second special issue of the journal on biological applications of X-ray absorption spectroscopy (BioXAS) is presented. The emphasis is on the study of metalloproteins in the context of structural genomics programmes (metallogenomics).


Subject(s)
Genomics , Metalloproteins/chemistry , Metalloproteins/genetics , Spectrum Analysis/methods , Protein Conformation , X-Rays
15.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 10): 1767-72, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14501116

ABSTRACT

Data acquisition from crystals of an icosahedral virus, cowpea mosaic virus (CPMV), was carried out to 2.8 A resolution under an elevated hydrostatic pressure of 330 MPa. This was the first example of a complex macromolecular assembly to be studied by high-pressure crystallography. The data were obtained from the ESRF ID30 beamline using a quasi-plane wave of ultrashort wavelength with a diamond anvil cell and an imaging-plate detector. The results of the high-pressure data analysis are given and are compared with those obtained under standard conditions, showing that the experimental procedures implemented are very efficient in terms of diffraction information collected per unit volume of crystal. These results suggest that the use of a quasi-parallel synchrotron radiation beam of ultrashort wavelength should also be considered for conventional macromolecular crystallography data collection.


Subject(s)
Comovirus/chemistry , Crystallography, X-Ray/methods , Crystallization , Data Collection/methods , Hydrostatic Pressure , Scattering, Radiation
16.
J Synchrotron Radiat ; 10(Pt 1): 1-3, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12511783

ABSTRACT

A special Issue of the Journal is presented, dedicated to biological applications of X-ray absorption spectroscopy (BioXAS) and examining the role of this technique in post-genomic biology. The Issue confirms that BioXAS has come of age and it can be expected to make a significant contribution in the structural genomics effort on metalloproteins, which are estimated to make up about 30% of proteins coded by genomes.


Subject(s)
Absorptiometry, Photon/methods , Genomics , Metalloproteins/chemistry , Metalloproteins/genetics
17.
J Synchrotron Radiat ; 10(Pt 1): 4-8, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12511784

ABSTRACT

A canonical structural genomics programme is being conducted at the Paris-Sud campus area on baker's yeast proteins. Experimental strategies, first results and identified bottlenecks are presented. The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.


Subject(s)
Fungal Proteins/chemistry , Genomics , Open Reading Frames/genetics , Cloning, Molecular , Escherichia coli/genetics , Fungal Proteins/genetics , Molecular Structure , Protein Conformation , Recombinant Proteins/chemistry , X-Ray Diffraction
18.
Structure ; 10(10): 1409-14, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12377126

ABSTRACT

The combined use of a diamond anvil cell and ultrashort-wavelength undulator radiation has allowed the collection of high-resolution diffraction data from protein and virus crystals submitted to hydrostatic pressures beyond 2 kbar. Crystals of cubic cowpea mosaic virus (CPMV) can be compressed to at least 3.5 kbar. Diffraction from CPMV crystals displaying an unusual disorder at atmospheric pressure was considerably enhanced by application of pressure. These experiments suggest that pressure may be used in some cases to improve order in crystals.


Subject(s)
Crystallography/methods , Proteins/chemistry , Viruses/chemistry , Pressure
19.
Methods Enzymol ; 276: 268-286, 1997.
Article in English | MEDLINE | ID: mdl-27799100
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