Single-pulse Laue diffraction, stroboscopic data collection and femtosecond flash photolysis on macromolecules

We review the time structure of synchrotron radiation and its use for fast time-resolved diffraction experiments in macromolecular photo-cycles using flash photolysis to initiate the reaction. The source parameters and optics for ID09 at ESRF are presented together with the phase-locked chopper and femtosecond laser. The chopper can set up a 900 Hz pulse train of 100 ps pulses from the hybrid bunch-mode and, in conjunction with a femtosecond laser, it can be used for stroboscopic data collection with both monochromatic and polychromatic beams. Single-pulse Laue data from Cutinase, a 22 kD lipolic enzyme, are presented which show that the quality of single-pulse Laue patterns is sufficient to refine the excited state(s) in a reaction pathway from a known ground state. The flash photolysis technique is discussed and an example is given for heme proteins. The radiation damage from a laser pulse in the femto and picosecond range can be reduced by triggering at a wavelength where the interaction is strong. We propose the use of microcrystals between 25–50 μm for efficient photolysis with femto and picosecond pulses. The performance of circular storage rings is compared with the predicted performance of an X-ray free electron laser (XFEL). The combination of micro beams, a gain of 105105 photons per pulse and an ultrashort pulse length of 100 fs is likely to improve pulsed diffraction data very substantially. It may be used to image coherent nuclear motion at atomic resolution in ultrafast uni-molecular reactions. © 1997 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87418/2/267_1.pd

Structural dynamics probed by X-ray pulses from synchrotrons and XFELs

This review focuses on how short X-ray pulses from synchrotrons and XFELs can be used to track light-induced structural changes in molecular complexes and proteins via the pump–probe method. The upgrade of the European Synchrotron Radiation Facility to a diffraction-limited storage ring, based on the seven-bend achromat lattice, and how it might boost future pump–probe experiments are described. We discuss some of the first X-ray experiments to achieve 100 ps time resolution, including the dissociation and in-cage recombination of diatomic molecules, as probed by wide-angle X-ray scattering, and the 3D filming of ligand transport in myoglobin, as probed by Laue diffraction. Finally, the use of femtosecond XFEL pulses to investigate primary chemical reactions, bond breakage and bond formation, isomerisation and electron transfer are discussed

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Strukturelle Dynamik von photosensitiven Proteinen

The thesis presented here describes the application of intense, pulsed X-rays from a synchrotron to the investigation of biochemical reactions triggered by light. The workincludes contributions to the instrumentation of a beamline and experiments with the proteins myoglobin, the photoactive yellow protein (PYP) and bacteriorhodopsin.The instrumentational work led to an improvement of the time resolution for pulsed Laue experiments from 10 ns to 100 ps. My contribution was the integration of three majornew components, a femtosecond laser system, a fast X-ray chopper and a heatload x-ray shutter, into the beamline control system. For laser/X-ray pump probe experiments, thetime delay can now be tuned under software control with picosecond precision. As a diagnostic to verify the photo-excitation of samples, I constructed a microsecond timeresolvedspectrometer with micro-focusing capabilities, which can measure the excitation degree of a sample online or offline. As part of an external collaboration, I contributed tothe construction of a ferntosecond time-resolved offline spectrometer. It was built to answer the question whether protein crystals can be excited with a focused femtosecondlaser to a sufficiently high degree without damage, and to identify the optimal triggering conditions.The laser triggering studies showed in the case of myoglobin that a protein crystal can be excited with ferntosecond laser pulses without disordering the crystalline structure orroducing photochemical damage. 100-ps time resolved Laue diffraction experiments on Myoglobin-CO show structural dynamics compatible with earlier nanosecondexperiments, but no clear indication for a CO docking site. A nanosecond time-resolved Laue experiment on PYP showed an early photocycle intermediate, with a photoisomerizedchromophore but little larger rearrangement in the protein. Although technically successful 100-ps time-resolved Laue diffraction experiments on PYP havebeen repeated several times, they have not yet given structural results. In the case of bacteriorhodopsin purple membrane, time-resolved powder diffraction with a choppedmonochromatic X-ray beam was used, accumulating thousands of images on the detector. In this way a millisecond time-resolved electron difference map for the wild-type M statewas obtained. The myoglobin and PYP experiments were collaborations with external workgroups

Die Drillkultur : ihre Vorzüge, ihre Rentabilität und ihre volkswirtschaftliche Bedeutung ; nebst einer speziellen Anleitung zur Stellung und Behandlung der Drills und Pferdehacken

hrsg. von C. J. Eisbein unter Mitw. des Ingenieurs F. Schott

Time-resolved methods in biophysics. 6. Time-resolved Laue crystallography as a tool to investigate photo-activated protein dynamics

When polychromatic X-rays are shined onto crystalline material, they generate a Laue diffraction pattern. At third generation synchrotron radiation sources, a single X-ray pulse of similar to 100 ps duration is enough to produce interpretable Laue data from biomolecular crystals. Thus, by initiating biological turnover in a crystalline protein, structural changes along the reaction pathway may be filmed by ultra-fast Laue diffraction. Using laser-light as a trigger, transient species in photosensitive macromolecules can be captured at near atomic resolution with sub-nanosecond time-resolution. Such pump-probe Laue experiments have now reached an outstanding level of sophistication and have found a domain of excellence in the investigation of light-sensitive proteins undergoing cyclic photo-reactions and producing stiff crystals. The main theoretical concepts of Laue diffraction and the challenges associated with time-resolved experiments on biological crystals are recalled. The recent advances in the design of experiments are presented in terms of instrumental choices, data collection strategy and data processing, and some of the inherent difficulties of the method are highlighted. The discussion is based on the example of myoglobin, a protein that has traversed the whole history of pump-probe Laue diffraction, and for which a massive amount of data have provided considerable insight into the understanding of protein dynamics

Picosecond time-resolved x-ray crystallography: probing protein function in real time

Abstract A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysisinduced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 ls. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally ''watch'' the protein as it executes its function. Published by Elsevier Inc

Real-time tracking of CO migration and binding in the ? and ? subunits of human hemoglobin via 150-ps time-resolved Laue crystallography

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (∼300 μs−1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2