Time-resolved Laue Crystallography Research Articles

Single pulse data collection with an X-ray chopper at in situ room temperature Laue crystallography beamline BL03HB

in situ room temperature Laue crystallography has gained increasing attention. This method is suitable for time-resolved Laue crystallography experiments to get crystal structure with only several single-frame data collections. To improve the diffraction efficiency of protein crystals at room temperature, a simple X-ray chopper combined with a fast shutter has been successfully developed and tested at in situ room temperature Laue crystallography beamline BL03HB of the Shanghai Synchrotron Radiation Facility (SSRF). This chopper is built with two aluminum blades. There are two windows on both blades, as well as an internal lead block. The width of windows for transmission of beam can be flexibly adjusted by a steel block coated with lead. The chopping frequency is controlled independently. The device allows chopping frequencies ranging from 0 to 10 Hz, and exposure times down to 100 μs can be obtained for our Laue diffraction experiment with the combination of a fast shutter. Moreover, a single pulse data collection strategy has been developed at BL03HB. The performance of our single pulse data collection method with the designed chopper for the improvement of data quality has been analyzed. Results show that the quality of diffraction data obtained from the single pulse data collection method is significantly improved, and the developed chopper is very suitable for room temperature Laue crystallography experiments at the synchrotron beamline with super bending magnet as light source.

Computational Study of PmHMGR Thiohemiacetal Breakdown Transition State

3-hydroxyl-3-methylglutaryl CoA reductase (HMGR) catalyzes the rate determining step in the isoprenoid pathway using two NADH molecules. Even though this enzyme is targeted by statins, the atomistic details of the mechanism are still unknown. This objective can be met by combination of computational biophysics and experimental validations through time-resolved Laue crystallography for all steps of the reaction. The catalysis of HMGR can be broken down into three major chemical transformations including two hydride transfers and a thiohemiacetal breakdown step. Moreover, since two cofactor molecules are required, a cofactor exchange process also occurs after the first hydride transfer and before the second hydride transfer. Thiohemiacetal is a known intermediate resulting from the first hydride transfer step. However, the product of the thiohemiacetal breakdown step hypothesized to be mevaldehyde, has not been experimentally validated. This theoretical study explores the atomic details of the enzyme and ligands for the thiohemiacetal breakdown step using the Pseudomonas mevalonii HMGR. Theozymes and QM/MM ONIOM models were used to identify the transition state were performed to reveal the catalytic residues. These models have revealed that the Nδ of His381 is involved in stabilizing the negative charge in the thiolate anion. Ser85 forms a hydrogen bonding network with the first amide of thiohemiacetal ligand, a function performed by His381 in the preceding step first hydride transfer. Transition state force fields (TSFF) using the quantum guided molecular mechanics (Q2MM) program will then be used to perform µsec timescale molecular dynamics simulations. This project focuses on not only developing computational biophysics methods for studying enzyme mechanisms but also uses time-resolved x-ray crystallography to obtain snapshots along the HMGR reaction pathway which involves multiple transformations of the ligand in the same active site, a large flap domain movement, and a cofactor exchange.

Probing Anisotropic Structure Changes in Proteins with Picosecond Time-Resolved Small-Angle X-ray Scattering

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals and suggest that the early time structural dynamics in the crystalline state carry over to proteins in solution.

Real-time tracking of CO migration and binding in the α and β 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, 2Å-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 100ps to 10μs. The proximity of the B site in the β subunit is about 0.25Å closer to its A binding site, and its kBA rebinding rate (∼300μs−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.

1P003 イェロープロテインの150ピコ秒時間分解能ラウエ構造解析(01A. 蛋白質:構造,ポスター,日本生物物理学会年会第51回(2013年度))

1P003 イェロープロテインの150ピコ秒時間分解能ラウエ構造解析(01A. 蛋白質:構造,ポスター,日本生物物理学会年会第51回(2013年度))

Activation Of Signaling Protein Is Visualized

Researchers have devised a way to watch a light-induced signaling protein as it functions, revealing new details about the way it is activated to do its job. The technique could make it possible to better understand step-by-step mechanisms of other signaling proteins, which control processes such as the movement of leaves toward light and vision in higher animals, and possibly the mechanisms of enzymes as well. In the study, the researchers used time-resolved Laue crystallography to take structural snapshots of bacterial photoactive yellow protein (PYP) in action. When PYP is activated by potentially harmful light frequencies, it provides signals that induce the bacteria to swim away. Using the Laue method, which harnesses polychromatic X-ray pulses to rapidly obtain structural information from protein crystals, the researchers captured PYP’s activation with near-atomic spatial resolution and 150-picosecond time resolution. Ultrafast biophysical chemistry specialist Philip A. Anfinrud of the National Inst...

Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography

To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90° out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.

Ultrafast Potential Energy Surface Softening of One-Dimensional Organic Conductors Revealed by Picosecond Time-Resolved Laue Crystallography

Time-resolved Laue crystallography has been employed to study the structural dynamics of a one-dimensional organic conductor (tetrathiafulvalene-p-chloranil) during photoexcitation in the regime of the neutral to ionic phase transition. Exciting this crystalline system with 800 nm 100 fs long optical pulses leads to ultrafast population of a structural intermediate as early as 50 ps after excitation with a lifetime of at least 10 ns. Starting from the neutral phase, this intermediate has been assigned as a precursor state toward the photoinduced population of the ionic phase. The observed intensity changes are significantly different from comparable equilibrium structures. The interpretation of this structural data is that the potential of this intermediate is being softened during its population in a dynamical process. The depopulation proceeds through thermal processes.

Myoglobin strikes back

Over the last half century, myoglobin (Mb) has been an excellent model system to test a number of concepts, theories, and new experimental methods that proved valuable to investigate protein structure, function, evolution, and dynamics. Mb's function, most often considered just an oxygen repository, has considerably diversified over the last 15 years, especially because it was shown to have a role in the biochemistry of quenching and synthesizing nitric oxide in the red muscle, thereby protecting the cell. To tackle protein's structural dynamics by innovative biophysical methods, Mb has been the best prototype; laser flash technology made it possible to obtain molecular movies by time-resolved Laue crystallography (with ps resolution). This approach unveiled the complexity of the energy landscape and the structural basis of the stretched interconversion between conformational substates of a protein.

Ligand Migration and Cavities within Scapharca Dimeric HbI: Studies by Time-Resolved Crystallo- graphy, Xe Binding, and Computational Analysis

As in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface.

The RATIO method for time-resolved Laue crystallography

A RATIO method for analysis of intensity changes in time-resolved pump-probe Laue diffraction experiments is described. The method eliminates the need for scaling the data with a wavelength curve representing the spectral distribution of the source and removes the effect of possible anisotropic absorption. It does not require relative scaling of series of frames and removes errors due to all but very short term fluctuations in the synchrotron beam.

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 approximately 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.

A Structural Pathway for Signaling in the E46Q Mutant of Photoactive Yellow Protein

In the bacterial photoreceptor photoactive yellow protein (PYP), absorption of blue light by its chromophore leads to a conformational change in the protein associated with differential signaling activity, as it executes a reversible photocycle. Time-resolved Laue crystallography allows structural snapshots (as short as 150 ps) of high crystallographic resolution (approximately 1.6 A) to be taken of a protein as it functions. Here, we analyze by singular value decomposition a comprehensive time-resolved crystallographic data set of the E46Q mutant of PYP throughout the photocycle spanning 10 ns-100 ms. We identify and refine the structures of five distinct intermediates and provide a plausible chemical kinetic mechanism for their inter conversion. A clear structural progression is visible in these intermediates, in which a signal generated at the chromophore propagates through a distinct structural pathway of conserved residues and results in structural changes near the N terminus, over 20 A distant from the chromophore.

Complexity of the structural dynamics of myoglobin: a study by time resolved Laue crystallography

Complexity of the structural dynamics of myoglobin: a study by time resolved Laue crystallography

Conformational dynamics of a myoglobin triple mutant observed by time-resolved Laue crystallography

Conformational dynamics of a myoglobin triple mutant observed by time-resolved Laue crystallography

Structure of the I1 early intermediate of photoactive yellow protein by FTIR spectroscopy.

To understand how proteins translate the energy of sunlight into defined conformational changes, we have measured the photocycle reactions of photoactive yellow protein (PYP) using time-resolved step scan Fourier transform infrared (FTIR) spectroscopy. Global fit analysis yielded the same apparent time constants for the reactions of the chromophore, the protonation changes of protein side chains and the protein backbone motions, indicating that the light cycle reactions are synchronized. Changes in absorbance indicate that there are at least four intermediates (I1, I1', I2, I2'). In the intermediate I1, the dark-state hydrogen bond from Glu 46 to the aromatic ring of the p-hydroxycinnamoyl chromophore is preserved, implying that the chromophore undergoes trans to cis isomerization by flipping, not the aromatic ring, but the thioester linkage with the protein. This excludes an I1 structural model proposed on the basis of time resolved Laue crystallography, but does agree with the cryotrapped structure of an I1 precursor.

Time‐resolved X‐ray crystallography: uncovering reaction intermediates in biochemical processes

X-ray crystallography plays a fundamental role in modern biochemistry and molecular biology, providing the structural basis for understanding the functionality of biological macromolecules. Indeed, biochemistry and molecular biology textbooks greatly benefit from the use of three-dimensional structures to address general concepts and to illustrate specific aspects of the biochemical processes. Structural analyses of model systems such as serine proteases are widely used to illustrate the molecular bases of key biochemical events, including protein-protein recognition, catalysis and enzyme regulation [ 1,2]. Moreover, the comparative analysis of the three-dimensional structures of deoxygenated and oxygenated hemoglobin, in the absence and presence of modulators, has provided a structure-based framework for a rational approach to complex phenomena such as allostery [3-51. Remarkably, about five new three-dimensional structures are deposited every day in the Protein Data Bank (http://www.rcsb.org/pdb), underlining the potentiality and the educational role of crystallography in biochemistry and molecular biology teaching. The biological activity of macromolecules is generally characterized by rapid (4 1 s) structural changes. However, due to the long time average represented by X-ray crystal structures (X-ray diffraction data are collected over intervals of several minutes to hours), the use of this technique in describing transient biochemical processes was, until recently, very limited. From this point of view, the development of time-resolved multiwavelength Laue crystallography was a major breakthrough, since it allows one to follow the time course of biochemical processes, through the related three-dimensional struc-

Towards automated Laue data processing: application to the choice of optimal X-ray spectrum.

Laue data reduction has now reached a level of sophistication that allows nearly automated processing to be performed. The software described enables complete reduction of the data with essentially no user intervention, making Laue processing almost as straightforward as monochromatic data processing. Interactive work is limited to the indexing of only one Laue pattern. More importantly, it is shown that the data quality is substantially enhanced when soft-limited predictions are used. Further improvement obtained by taking advantage of the structure-factor amplitudes from a known closely related structure is described. To determine the most suitable type of insertion device to be used for time-resolved Laue crystallography, the technique described was applied to Laue data sets collected from photoactive yellow protein under identical conditions but with three different insertion devices: a wiggler, a broad-bandpass undulator and a single-line undulator. Although the optimal choice may ultimately be dictated by sample parameters (such as mosaic spread) and by the type of experiment (repeatable or non-repeatable reactions), the results here show that the use of single-line undulators will generally yield by far the best compromise between data quality, acquisition time and radiation damage.

A 2D smart pixel detector for time-resolved crystallography

A smart pixel detector is being developed for time-resolved crystallography for biological and material science applications. Using the pixel detector presented here, the Laue method will enable the study of the evolution of structural changes that occur within a protein as a function of time. The x-ray pixellated detector is assembled to the integrated circuit through a bump bonding process. Within a pixel size of 150×150 mm2, a low-noise preamplifier-shaper, a discriminator, a 3-bit counter and the readout logic are integrated. The readout, based on the column architecture principle, will accept hit rates above 5×108/cm2/s with a maximum hit rate per pixel of 1 MHz. This detector will allow time-resolved Laue crystallography to be performed in a frameless operation mode, without dead time. Target specifications, architecture, and preliminary results on a 8×8 prototype are presented.

Time-resolved Laue crystallography: application to the photocycle of photoactive yellow protein

Time-resolved Laue crystallography: application to the photocycle of photoactive yellow protein