Ultrafast Biophysical Studies of Biomolecules at the APS

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In this report, we focus on time-resolved X-ray studies that employ the pump-probe method. Briefly, a laser pulse (pump) photoactivates or thermally excites a biomolecule, after which a suitably delayed X-ray pulse (probe) passes through the sample and records a diffraction or scattering pattern on a 2D detector. Thanks to significant capital equipment investments made by the NIH (over $1.2M since 2006), we have developed the ability to pursue studies of biomolecules on the BioCARS 14-IDB beamline via both time-resolved Laue crystallography and time-resolved SAXS/WAXS. Time-resolved Laue crystallography takes advantage of a polychromatic X-ray pulse, which can produce thousands of reflections in a single shot, and boosts substantially the rate at which time-resolved diffraction data can be acquired. The information needed to determine the protein's structure is encoded in the relative intensities of the diffraction spots observed. Since the structural information contained in a single Laue diffraction image is incomplete, repeated measurements at multiple crystal orientations are required to produce a complete set of data. Prior studies have generally required a small number of very large, homogeneous crystals, which has limited the application of this methodology to a handful of proteins. The impact of time-resolved Laue crystallography would be boosted significantly if we succeeded in developing methods capable of acquiring high signal-to-noise ratio diffraction images from a large number of relatively small crystals (30-35 microns), rather than small number of large crystals. To that end, we continue our efforts to develop novel microfluidic methods for growing crystals and delivering them in a fashion that can be automated. Briefly, we have developed an alternating drop microfluidic mixer that has been used to grow more than 1000 uniformly sized lysozyme crystals ( 30-35 microns) in a 1-m long glass capillary. A microfluidic crystal delivery system based on a home-built multi-axis syringe-pump tower is being developed with an aim to automate crystal delivery to the BioCARS 14-IDB beamline. This effort also includes the development of a high-speed diffractometer capable of rapidly and precisely positioning crystals at the intersection of the laser and X-ray beams. While much progress has been made, more work remains to be done. Our aim is to automate the acquisition of X-ray diffraction images from thousands of crystals without user intervention, and do so repeatedly. Time-resolved Laue crystallography, as its name implies, can only be performed on crystalline samples. The intermolecular forces that maintain crystalline order constrain large amplitude conformational motion, and this loss of flexibility may perturb or even inhibit the function of a protein. Though Laue crystallography stands alone in its ability to track structural changes in proteins on ultrafast time scales with near-atomic spatial resolution, it is crucial to also study structural dynamics of biomolecules in solution where the full range of conformational motion is permitted. Without external alignment forces, biomolecules in solution are randomly oriented, and the structural information contained in their orientationally-averaged diffuse scattering pattern is one dimensional. Nevertheless, it is well known that the SAXS region of the diffraction pattern reports on the size and shape of the biomolecule, while the WAXS region is sensitive to secondary and tertiary structure. Time-resolved SAXS/WAXS scattering patterns therefore provide 'fingerprints' that can be correlated with protein structure via molecular models, and can assess which models best describe reaction pathways in solution. Our time-resolved SAXS/WAXS diffractometer currently employs a secondary K-B mirror pair to focus the X-ray beam onto the sample capillary with independent control of the the vertical and horizontal dimensions, a very small beamstop (0.51 mm diameter), and a large area (340x340 mm), high-speed (up to 10 Hz) X-ray detector. With the sample-detector distance set at 185.8 mm, scattering data can be acquired over a broad range of q (momentum transfer) spanning 0.02 to 5.4 inverse Angstroms, which corresponds to spatial resolution below 1.2 Angstroms. To mitigate the adverse effects of radiation damage during X-ray exposure, the capillary containing the protein solution is rapidly translated over a 20-mm span using a home-built, high-speed diffractometer that is based on 1-micron resolution linear motor translation stages capable of more than 1-g acceleration. Thanks to a closed-loop circulation system, about 150 microliter of protein solution is sufficient to acquire a high signal-to-noise ratio data set. With our recently improved capillary holder and high-precision temperature controller, we are able to characterize structural changes over a broad range of temperatures spanning from approximately -16 to 120 degrees Celsius. Moreover, thanks to the relatively small x-ray spot size that can be generated with the secondary K-B mirror pair, it is possible to focus a 1-mJ infrared laser pulse down to a dimension small enough to heat samples in a glass capillary by more than 20 degrees Celsius. When setting the sample temperature just below its unfolding temperature, this magnitude T-jump is sufficient to trigger unfolding of the biomolecule, and allows us to investigate the dynamics of protein unfolding with unprecedented spatial resolution. The time-resolution achieved is currently limited by the duration of the infrared laser pulse, which is about 5 ns. In a collaboration with James Fraser of UCSF, we pursued a time-resolved study of conformational changes induced in cyclophilin A, a dynamic enzyme, following a temperature jump. This experiment was quite challenging due to the modest amplitude of the structural changes induced by the temperature jump. Nevertheless, we put significant effort into minimizing systematic error in the measurements and repeating them a large number of times. We succeeded in capturing functional intramolecular protein dynamics on the microsecond timescale, and showed that cyclophilin A displays rich dynamics following a temperature jump, with the resulting time-resolved signal allowing us to assess the kinetics of conformational changes. Two relaxation processes were resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results were published in Nature Chemistry. We have used our ever-improving time-resolved SAXS/WAXS methodology to study a wide variety of proteins as well as RNA hairpins, and are developing novel data analysis tools that promise to unveil at an unprecedented level of detail information about secondary structure, protein-protein interactions, and the time scale at which structural changes can occur. As our time-resolved SAXS/WAXS methodology becomes more precise and easier to use, we expect it to become an ever more important complement to time-resolved Laue studies and time-resolved optical spectroscopy studies of biomolecules, and will help provide a structural basis for understanding how biomolecules function.