Thomas Ursby

Photolysis of the carbon monoxide complex of myoglobin : Nanosecond time-resolved crystallography

The biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.

Helix deformation is coupled to vectorial proton transport in the photocycle of bacteriorhodopsin

A wide variety of mechanisms are used to generate a proton-motive potential across cell membranes, a function lying at the heart of bioenergetics. Bacteriorhodopsin, the simplest known proton pump, provides a paradigm for understanding this process. Here we report, at 2.1 Å resolution, the structural changes in bacteriorhodopsin immediately preceding the primary proton transfer event in its photocycle. The early structural rearrangements propagate from the proteins core towards the extracellular surface, disrupting the network of hydrogen-bonded water molecules that stabilizes helix C in the ground state. Concomitantly, a bend of this helix enables the negatively charged primary proton acceptor, Asp 85, to approach closer to the positively charged primary proton donor, the Schiff base. The primary proton transfer event would then neutralize these two groups, cancelling their electrostatic attraction and facilitating a relaxation of helix C to a less strained geometry. Reprotonation of the Schiff base by Asp 85 would thereby be impeded, ensuring vectorial proton transport. Structural rearrangements also occur near the proteins surface, aiding proton release to the extracellular medium.

The crystallography beamline I711 at MAX II.

A new X-ray crystallographic beamline is operational at the MAX II synchrotron in Lund. The beamline has been in regular use since August 1998 and is used both for macro- and small molecule diffraction as well as powder diffraction experiments. The radiation source is a 1.8 T multipole wiggler. The beam is focused vertically by a bendable mirror and horizontally by an asymmetrically cut Si(111) monochromator. The wavelength range is 0.8-1.55 A with a measured flux at 1 A of more than 10(11) photons s(-1) in 0.3 mm x 0.3 mm at the sample position. The station is currently equipped with a Mar345 imaging plate, a Bruker Smart 1000 area CCD detector and a Huber imaging-plate Guinier camera. An ADSC 210 area CCD detector is planned to be installed during 2000.

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.

Feasibility and Realization of Single-Pulse Laue Diffraction on Macromolecular Crystals at ESRF.

Laue diffraction patterns with an exposure time of ca 60 ps have been acquired at the European Synchrotron Radiation Facility (ESRF) on protein crystals by using the single-bunch mode of the storage ring. A 10 ns laser pulse initiating photodissociation was synchronized with the X-ray pulse. The potential for a quantitative detection of conformational changes in proteins on the nanosecond timescale with this technique is demonstrated using the example of carbonmonoxymyoglobin, from simulations and real data. The instrumental aspects of the experiment (highly intense X-ray beam, fast shutter system, Laue camera, detector, laser apparatus and synchronization technique) are emphasized.

The macromolecular crystallography beamline I911-3 at the MAX IV laboratory

The updated macromolecular crystallography beamline I911-3 at the MAX II storage ring is described.

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.

Spectroscopic Characterization of Bacteriorhodopsin's L‐intermediate in 3D Crystals Cooled to 170 K¶

Spectra are presented from a single 3D microcrystal of bacteriorhodopsin (bR) cooled to 170 K under various illumination conditions. This set is necessary and sufficient to assign the relevant crystal reference spectra. A spectral decomposition of the difference spectrum obtained following the trapping protocol of Royant et al.(2000) (Nature406, 645–648) is given, confirming that the low temperature L‐intermediate was the species that dominated the structural rearrangements previously reported. Smaller contributions from the K and M spectral intermediates are also quantified. Mechanistic insights derived from the X‐ray structures of the early bR intermediates are discussed.

Time-resolved structures of hydroxymethylbilane synthase (Lys59Gln mutant) as it is loaded with substrate in the crystal determined by Laue diffraction

The structure of the catalytically active, reduced, form of the enzyme hydroxymethylbilane synthase (HMBS, Lys59Gln mutant) has been studied by Laue diffraction as the substrate, porphobilinogen (PBG), was fed to an immobilised crystal in a flow cell. Laue data at short time-scale time points (i.e. 1, 2, 4 and 8 min) were measured using several crystals and then averaged. Longer time-point data sets (i.e. 25 min ± 7 min and 2 h 23 min ± 9 min) were measured from individual crystals. All data sets benefited from using rapid exposures on ESRF ID09 (≈1 ms) and the fast duty cycle ESRF II CCD (readout time 8s). In one case, the substrate supply to a crystal in the flow cell was stopped at 4 h and monochromatic data collected at 12 h ± 30 min on ESRF BM14 (i.e. about 8 h after the substrate supply was stopped). Structural analysis of these data sets at all the time points was undertaken commencing with rigid-body refinement based upon the molecular model of the active, reduced, enzyme. The rigid-body refinement showed that rotational and translational movements of individual domains of the protein are less than 0.7° and 0.2 A, respectively in the crystal with respect to the wild-type active form model. Moreover, difference Fourier maps at different time points versus the wild-type reduced form were calculated based upon the calculated phases from the wild-type reduced form model. These maps show that at 8 min, 25 min and 2 h, extended electron density appears in the active site region. This electron density is not visible in the 12 h case. Detailed structural refinement on the 2 h data, for which the extra electron density is most prominent, allowed an improved omit-type difference map to be calculated. This shows electron density in the active site adjacent and above the side-chain of Asp84, which plays a pivotal role throughout the catalytic reaction cycle. The density peak commences at the cofactor C2 ring (oxidised form) position (earlier proposed as a binding site for PBG). It then extends up towards Arg149, past Arg155 (residues whose mutation causes build up of ES1 and ES4 intermediate enzyme–substrate complexes, respectively) and out towards the open solvent channel of the crystal.

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 Physics