Biomedical subjects

Dominique Bourgeois

Publications and source records attributed to Dominique Bourgeois.

12 recordsLinked to original sources

PubMed2006

X-ray radiation-induced damage in DNA monitored by online Raman.

High-quality Raman spectra have been collected on single DNA crystals kept at 100 K in a flow of cold nitrogen gas prior, during and after macromolecular crystallography (MX) data collection. The non-symmetric bending vibration for C-Br bonds in 8-bromo-2'-deoxyguanosine has been unambiguously identified at 293 cm(-1) in the non-resonant Raman spectra. The breakage of this bond could be monitored by the continuous recording of Raman spectra during X-ray exposure, and its decay versus dose could be directly correlated with the loss of the bromide definition within the electron-density maps as determined with MX.

Computer Simulation↗

PubMed2006

Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography.

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase ( approximately ns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.

Carbon Monoxide↗

PubMed2006

UV laser-excited fluorescence as a tool for the visualization of protein crystals mounted in loops.

Structural proteomics has promoted the rapid development of automated protein structure determination using X-ray crystallography. Robotics are now routinely used along the pipeline from genes to protein structures. However, a bottleneck still remains. At synchrotron beamlines, the success rate of automated sample alignment along the X-ray beam is limited by difficulties in visualization of protein crystals, especially when they are small and embedded in mother liquor. Despite considerable improvement in optical microscopes, the use of visible light transmitted or reflected by the sample may result in poor or misleading contrast. Here, the endogenous fluorescence from aromatic amino acids has been used to identify even tiny or weakly fluorescent crystals with a high success rate. The use of a compact laser at 266 nm in combination with non-fluorescent sample holders provides an efficient solution to collect high-contrast fluorescence images in a few milliseconds and using standard camera optics. The best image quality was obtained with direct illumination through a viewing system coaxial with the UV beam. Crystallographic data suggest that the employed UV exposures do not generate detectable structural damage.

Crystallization↗

PubMed2006

The 1.6A X-ray structure of the unusual c-type cytochrome, cytochrome cL, from the methylotrophic bacterium Methylobacterium extorquens.

The structure of cytochrome cL from Methylobacterium extorquens has been determined by X-ray crystallography to a resolution of 1.6 A. This unusually large, acidic cytochrome is the physiological electron acceptor for the quinoprotein methanol dehydrogenase in the periplasm of methylotrophic bacteria. Its amino acid sequence is completely different from that of other cytochromes but its X-ray structure reveals a core that is typical of class I cytochromes c, having alpha-helices folded into a compact structure enclosing the single haem c prosthetic group and leaving one edge of the haem exposed. The haem is bound through thioether bonds to Cys65 and Cys68, and the fifth ligand to the haem iron is provided by His69. Remarkably, the sixth ligand is provided by His112, and not by Met109, which had been shown to be the sixth ligand in solution. Cytochrome cL is unusual in having a disulphide bridge that tethers the long C-terminal extension to the body of the structure. The crystal structure reveals that, close to the inner haem propionate, there is tightly bound calcium ion that is likely to be involved in stabilization of the redox potential, and that may be important in the flow of electrons from reduced pyrroloquinoline quinone in methanol dehydrogenase to the haem of cytochrome cL. As predicted, both haem propionates are exposed to solvent, accounting for the unusual influence of pH on the redox potential of this cytochrome.

Amino Acid Sequence↗

PubMed2005

The crystal structure of Mycobacterium tuberculosis thymidylate kinase in complex with 3'-azidodeoxythymidine monophosphate suggests a mechanism for competitive inhibition.

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis thymidylate kinase (TMPK(Mtub)) catalyzes the ATP-dependent phosphorylation of deoxythymidine 5'-monophosphate (dTMP). Essential to DNA replication, this enzyme represents a promising target for developing new drugs against TB, because the configuration of its active site is unique within the TMPK family. Indeed, it has been proposed that, as opposed to other TMPKs, catalysis by TMPK(Mtub) necessitates the transient binding of a magnesium ion coordinating the phosphate acceptor. Moreover, 3'-azidodeoxythymidine monophosphate (AZTMP) is a competitive inhibitor of TMPK(Mtub), whereas it is a substrate for human and other TMPKs. Here, the crystal structures of TMPK(Mtub) in complex with deoxythymidine (dT) and AZTMP were determined to 2.1 and 2.0 A resolution, respectively, and suggest a mechanism for inhibition. The azido group of AZTMP perturbs the induced-fit mechanism normally adopted by the enzyme. Magnesium is prevented from binding, and the resulting electrostatic environment precludes phosphoryl transfer from occurring. Our data provide a model for drug development against tuberculosis.

Binding Sites↗

PubMed2005

Advances in kinetic protein crystallography.

Many proteins function in the crystalline state, making crystallography a tool that can address mechanism, as well as structure. By initiating biological turnover in the crystal, transient structural species form, which may be filmed by Laue diffraction or captured by freeze-trapping methods. Laue diffraction has now reached an unprecedented level of sophistication and has found a 'niche of excellence' in the study of cyclic, ultra-fast, light-triggered reactions. Trapping methods, on the other hand, are more generally applicable, but require care to avoid artifacts. New strategies have been developed and difficulties such as radiation damage have received particular attention. Complementary methods--mainly UV/visible single-crystal spectroscopy--have proven essential to design, interpret and validate kinetic crystallography experiments.

Animals↗

PubMed2004

Structural basis for the mechanism of Ca(2+) activation of the di-heme cytochrome c peroxidase from Pseudomonas nautica 617.

Cytochrome c peroxidase (CCP) catalyses the reduction of H(2)O(2) to H(2)O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca(2+) and was refined at 2.2 A resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca(2+) dependent and was crystallized at pH 5.3 and refined at 2.4 A resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

Amino Acid Sequence↗

PubMed2004

Structure of superoxide reductase bound to ferrocyanide and active site expansion upon X-ray-induced photo-reduction.

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site.

Bacterial Proteins↗

PubMed2004

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals.

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.

Acetylcholinesterase↗

PubMed2003

Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography.

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron-ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-A resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100-300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (< or =3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function.

Amino Acid Substitution↗

PubMed2003

Characterization of photocycle intermediates in crystalline photoactive yellow protein.

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.

Bacterial Proteins↗

PubMed2002

Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals.

Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s(-1)) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in flash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100-150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryo-photolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.

Adenosine Triphosphate↗