33 research outputs found
A microspectrophotometer for UV–visible absorption and fluorescence studies of protein crystals
From Time-Averaged to Time-Resolved Crystallography: Studies on Superoxide Dismutase and Myoglobin
We have studied protein motions with nanosecond time-resolved crystallography. The release of carbon monoxide from its complex with myoglobin (MbCO) was triggered by nanosecond laser pulses. X-ray data were collected with time delays ranging from 4 ns to 1.9 ms using the Laue method, at the European Synchrotron Radiation Facility in Grenoble. For the first time point, X-ray pulses of 150 ps duration were used. Electron difference maps show the release of the CO and the subsequent motion of the iron out of the haem plane. In the 4 ns difference map, a positive density feature is found at a position coinciding with that of an intermediate docking site seen at low temperature. The following time points show the rebinding of the CO, and also indications of protein relaxations extending further than 4 ns. This is the first time-resolved protein crystallography experiment with nanosecond time-resolution yielding structural results. We have developed instrumentation and methods for time-resolved protein crystallography, including a method to reduce the noise in electron density difference maps from poorly accurate data. The method is also applicable to other cases where the signal to noise ratio is low. The crystal structure of iron superoxide dismutase (SOD) from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined to 2.3 Å resolution by molecular replacement. The structure revealed an increased number of inter-subunit ion-pairs in a compact tetramer. We suggest this to be important for the thermostability. However, the general fold is found to be similar to other known iron- or manganese SOD structures. In addition, the electron density maps revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site
Bent Diamond Crystals and Multilayer Based Optics at the new 5-Station Protein Crystallography Beamline ‘Cassiopeia’ at MAX-lab
A new 5-station beamline for protein crystallography is being commissioned at the Swedish synchrotron light source MAX-II at Lund University. Of the 2K/gamma = 14 mrad horizontal wiggler fan, the central 2 mrad are used and split in three parts. The central 1 mrad will be used for a station optimized for MAD experiments and on each side of the central fan, from 0.5 mrad to 1 mrad, there are two fixed energy stations using different energies of the same part of the beam. These, in total five stations, can be used simultaneously and independently for diffraction data collection. The two upstream monochromators for the side stations are meridionally bent asymmetric diamond(111) crystals in Laue transmission geometry. The monochromators for the downstream side stations are bent Ge(111) crystals in asymmetric Bragg reflection geometry. Curved multilayer mirrors inserted in the monochromatic beams provide focusing in the vertical plane. The first side station is under commissioning, and a preliminary test protein data set has been collected. ©2004 American Institute of Physic
Vibrational stability of a cryocooled horizontal double-crystal monochromator
The vibrational stability of a horizontally deflecting double-crystal monochromator (HDCM) is investigated. Inherently a HDCM will preserve the vertical beam stability better than a `normal' vertical double-crystal monochromator as the vibrations of a HDCM will almost exclusively affect the horizontal stability. Here both the relative pitch vibration between the first and second crystal and the absolute pitch vibration of the second crystal are measured. All reported measurements are obtained under active cooling by means of flowing liquid nitrogen (LN2). It is found that it is favorable to circulate the LN2 at high pressures and low flow rates (up to 5.9 bar and down to 3 l min(-1) is tested) to attain low vibrations. An absolute pitch stability of the second crystal of 18 nrad RMS, 2-2500 Hz, and a relative pitch stability between the two crystals of 25 nrad RMS, 1-2500 Hz, is obtained under cryocooling conditions that allow for 1516 W to be adsorbed by the LN2 before it vaporizes
A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals
Absorption microspectrophotometry has been shown to be of considerable help to probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. Absorption spectra contribute to the proper interpretation of crystallographic structures, especially when transient intermediate states are studied. Here it is shown that fluorescence microspectrophotometry might also be used for such purposes if endogenous fluorophores are present in the macromolecule or when exogenous fluorophores are added and either bind to the protein or reside in the solvent channels. An off-line microspectrophotometer that is able to perform low-temperature absorption and fluorescence spectroscopy on crystals mounted in cryo-loops is described. One-shot steady-state emission spectra of outstanding quality were routinely collected from several samples. In some cases, crystals with optical densities that are too low or too high for absorption studies can still be tackled with fluorescence microspectrophotometry. The technique may be used for simple controls such as checking the presence, absence or redox state of a fluorescent substrate/co-factor. Potential applications in the field of kinetic crystallography are numerous. In addition, the possibility to probe key physico-chemical parameters of the crystal, such as temperature, pH or solvent viscosity, could trigger new studies in protein dynamics
Mycobacterium tuberculosis thymidylate kinase: structural studies of intermediates along the reaction pathway.
International audienceMycobacterium tuberculosis TMP kinase (TMPK(Mtub)) represents a promising target for developing drugs against tuberculosis because the configuration of its active site is unique in the TMPK family. To help elucidate the phosphorylation mechanism employed by this enzyme, structural changes occurring upon binding of substrates and subsequent catalysis were investigated by protein crystallography. Six new structures of TMPK(Mtub) were solved at a resolution better than 2.3A, including the first structure of an apo-TMPK, obtained by triggering catalysis in a crystal of a TMPK(Mtub)-TMP complex, which resulted in the release of the TDP product. A series of snapshots along the reaction pathway is obtained, revealing the closure of the active site in going from an empty to a fully occupied state, suggestive of an induced-fit mechanism typical of NMPKs. However, in TMPK(Mtub) the LID closure couples to the binding with an unusual location for a magnesium ion coordinating TMP in the active site. Our data suggest strongly that this ion is required for catalysis, acting as a clamp, possibly in concert with Arg95, to neutralise electrostatic repulsion between the anionic substrates, optimise their proper alignment and activate them through direct and water-mediated interactions. The 3'-hydroxyl moiety of TMP, critical to metal stabilisation, appears to be a target of choice for the design of potent inhibitors. On the other hand, the usual NTP-bound magnesium is not seen in our structures and Arg14, a P-loop residue unique to TMPK(Mtub), may take over its role. Therefore, TMPK(Mtub) seems to have swapped the use of a metal ion as compared with e.g. human TMPK. Finally, TTP was observed in crystals of TMPK(Mtub), locked by Arg14, thus providing a structural explanation for the observed inhibitory effect of TTP putatively involved in a mechanism of feedback regulation of the enzymatic activity
Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals
International audienceCaged 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
Iron superoxide dismutase from the archaeon Sulfolobus sulfataricus : Analysis of structure and thermostability.
The crystal structure of superoxide dismutase (SOD) from the hyper thermophile Sulfolobus solfataricus has been determined at 2.3 Å resolution by molecular replacement and refined to a crystallographic R-factor of 16.8 % (Rfree 19.8 %). The crystals belong to the space group C2 (a = 76.3 Å, b = 124.3 Å,c = 60.3 Å, β = 128.8°) with two identical monomers in the asymmetric unit. The monomer has a molecular weight of 24 kDa and consists of 210 amino acid residues of which 205 are visible in the electron density map. The overall fold of the monomer of S. solfataricus SOD is similar to that of the other known Fe or Mn-SODs. S. solfataricus SOD forms a very compact tetramer of a type similar to that of SOD from the hyperthermophile Aquifex pyrophilus. Both structures show an elevated number of inter-subunit ion-pairs compared with the mesophilic SOD from Mycobacterium tuberculosis and the thermophilic SOD from Thermus thermophilus. However, in contrast to the A. pyrophilus SOD structure, the number of intra-subunit ion-pairs as well as inter-subunit hydrogen bonds is not higher than in the compared mesophilic and thermophilic SOD structures. The electron density also revealed an unexpected and unusual covalent modification of a conserved tyrosine in the active site. Its involvement in the specific activity of the enzyme is discussed