Ultrafast Biophysical Studies of Proteins: Time-resolved Laue crystallography

Anfinrud, Philip

Abstract

Our dream of watching a protein function in real time with 150 ps time resolution and near-atomic spatial resolution was first realized in 2003 using picosecond time-resolved Laue crystallography, an experimental methodology developed by the Anfinrud group at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. To advance this capability further, we initiated in 2005 a major effort to develop the infrastructure required to pursue picosecond time-resolved X-ray science on the BioCARS 14-IDB beamline at the Advanced Photon Source (APS) in Argonne, IL. That effort, which has been quite successful, is documented in a separate report. We acquire time-resolved Laue diffraction images using the pump-probe method. Briefly, a laser pulse (pump) photoactivates a protein crystal, after which a suitably delayed X-ray pulse (probe) passes through the crystal and records its diffraction pattern on a 2D detector. Because we use a polychromatic X-ray pulse, we capture thousands of reflections in a single image without having to rotate the crystal. This Laue approach to crystallography boosts substantially the rate at which time-resolved diffraction data can be acquired. The information needed to determine the proteins structure is encoded in the relative intensities of the diffraction spots observed. However, the structural information contained in a single diffraction image is incomplete, requiring repeated measurements at multiple crystal orientations to produce a complete set of data. We continue to develop TReX, an in-house software package designed to analyze time-resolved Laue data, and are working on TReX-II, a major update to this software package that employs a ratio method for data processing. The ratio method is based upon back-to-back diffraction images acquired with (ON) and without (OFF) a laser pump pulse. By merging ION/IOFF ratios of integrated spot intensities for each indexed reflection, instead of intensity differences, errors arising from image scaling and wavelength normalization are mitigated. After merging, the ratios are easily converted to structure factor amplitude differences, which are needed for structure refinement. In November 2013, we attempted a femtosecond time-resolved X-ray diffraction study of a heme protein at the LCLS in Stanford, California. The LCLS is the world's first X-ray free electron laser, and produces intense X-ray pulses shorter than 100 fs in duration (<10-15 s). For this study, we designed a novel high-speed X-ray diffractometer and installed it on the XPP beam line at the LCLS. This study proved quite challenging: we attempted to use a high-speed X-ray detector for the first time; we had to develop beamline control software capable of synchronizing our diffractometer with the X-ray laser used to probe the protein crystal; and had to synchronize the firing of the optical laser used to photo excite the protein crystal. Moreover, we had to develop a protocol for scanning the protein crystal through the X-ray beam to find its edge to a precision of at least 10 m. Though we succeeded in getting the infrastructure functioning properly, there wasn't sufficient beam time left to acquire the amount of data needed to produce a femtosecond time-resolved movie of the structural changes occurring in the protein crystal. We did, however, succeed in demonstrating a photocrystallography chip that localizes protein crystals in a two-dimensional array of holes. When loaded at high density, this approach to sample handling allow rapid acquisition of time-resolved diffraction images. Unfortunately, drift of the X-ray beam position degraded the intensity transmitted through the X-ray slits during the period over which data from the photocrystallography chip were acquired, and the diffraction patterns were too weak and too few to solve the structure of the earliest intermediates generated by laser photolysis. Nevertheless, the data indicated laser-induced spot intensity changes that unambiguously arose from a structural change in the protein. This result was published in Structural Dynamics 2, 054302 (2015). The best time-resolved diffraction data acquired in the past has come from high quality, large protein crystals. It has always been a challenge to secure a suitable number of large, high quality crystals for those studies. To take our time-resolved Laue crystallography studies to the next level, it is crucial to develop the infrastructure needed to generate movies of structural changes of proteins using much smaller crystals. For a variety of reasons, we are aiming for crystals that are about 35 microns in size. Due to radiation damage, the amount of data that can be acquired from a crystal of that size is limited. Therefore a large number will be required to attain the same information content available from a few large ones. The time required to mount and align a protein crystal in the hutch and then interlock the hutch so data can be acquired would be prohibitive with such small crystals. Hence, we are working on a novel micro-fluidic approach for not only growing crystals of a uniform size, but also for delivering and centering them at the intersection of the laser and X-ray beams on the BioCARS beamline. Though much work remains, the approaches being developed are quite promising. By capturing the structure and temporal evolution of key reaction intermediates, time-resolved Laue crystallography provides an unprecedented view into the relations between protein structure, dynamics, and function. Being able to acquire such detailed information in a variety of protein systems is crucial to properly assess the validity of theoretical and computational approaches in biophysics, and to unveil reaction pathways that are at the heart of biological functions.