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 highly 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 avoided. After merging, the ratios are easily converted to structure factor amplitude differences, which are needed for structure refinement. We have used photoactive yellow protein (PYP) as a model system for understanding how signaling proteins function. PYP is a 14-kD water-soluble blue-light receptor that, upon absorbing a single photon, produces a signaling state that causes a purple sulfur bacterium, Halorhodospira halophile, to swim away from blue light. The chromophore in PYP is p-coumaric acid (pCA), which is covalently linked to the Cys69 residue via a thioester bond. Its C2=C3 double bond is trans in the ground state, but upon absorbing a photon, is converted to cis with modest quantum efficiency. This photoisomerization event triggers a sequence of structural changes that involve spectroscopically red-shifted (pR) and blue-shifted (pB) intermediates, the last of which corresponds to the putative signaling state. We have used time-resolved Laue crystallography to investigate the structures of the intermediates produced as this protein progresses through its fully reversible photocycle, and published these results in the Nov 12 issue of PNAS. This effort proved quite challenging. The PYP crystals proved to be radiation sensitive, which limited the number of diffraction images that could be acquired from a single protein crystal. To mitigate the adverse effects of radiation damage, we spread the x-ray/laser dose over the entire length of large crystals, and acquired complete time series at several different orientations with each crystal. With numerous crystals, we achieved sufficient completeness to generate high-resolution structures over a complete time series spanning 10 decades of time with 150 ps time resolution. We also developed a real-space global analysis method to characterize the structures of intermediates represented in the experimental data, and determine the kinetics for their interconversion. The kinetic rate parameters used to define the time-dependent populations of putative intermediates are refined by non-linear least squares while the electron density difference maps for the corresponding intermediates are determined by linear least squares. We start with a plausible kinetic model whose corresponding rate equations account not only for the first-order processes for structure interconversion, but also for the rate of photoactivation, which depends upon the instrument response function, i.e., the convolution of the laser and x-ray pulses. The ability to properly account for the experimental instrument response function is crucial for accurate determination of lifetimes approaching its width, which in this case is 150 ps. We found that four intermediates were required to account for our experimental time-resolved electron density maps, the first three of which are red-shifted species, and the last of which is a blue-shifted species. Hence, we denote these four intermediates pR0, pR1, pR2, and pB0. Structures similar to pR1, pR2, and pB0 have been reported before, but pR0 is novel. The simplest possible kinetic model connecting these intermediates was found to be sequential with a reversible pR0 to pR1 transition, and with a pR2 to pB0 transition that short circuits to the ground pG state approximately half the time. This simple model accounts for the experimental electron density maps with high fidelity. The novel pR0 structure unveiled through this work corresponds to a highly strained cis intermediate that launches the PYP photocycle. The pCA carbonyl in pR0 is oriented 90 out of the plane of the phenolate and appears to be locked in this twisted conformation by the hydrogen bond between this carbonyl and the Cys69 backbone nitrogen. In a collaboration with Dr. Gerhard Hummer, this unusual conformation was examined by Density Functional Theory (DFT) calculations and found to be chemically plausible. Moreover, the x-ray refined and DFT-optimized structures were found to be in excellent agreement for all other intermediates as well, thereby cross-validating these structures and confirming the plausibility of our pR0 structure. The time-resolved structural evolution observed in this study, spanning ten decades of time, unveiled a simple, stepwise structural progression of PYP conformations toward a long-lived pB0 state, with each transition characterized at an unprecedented level of detail. The highly contorted pR0 state provides a visual clue regarding the conformational gymnastics that must accompany trans/cis isomerization in a highly constrained protein environment. This study illustrates how a protein can employ hydrogen bonding networks, gated water penetration, and strain to steer the direction of structural transitions in a fashion that facilitates its target function. By capturing the structure and temporal evolution of key reaction intermediates, picosecond time-resolved Laue crystallography provides an unprecedented view into the relations between protein structure, dynamics, and function. Such detailed information is crucial to properly assess the validity of theoretical and computational approaches in biophysics. By combining incisive experiments and theory, we move closer to resolving reaction pathways that are at the heart of biological functions.
|Kaila, Ville R I; Schotte, Friedrich; Cho, Hyun Sun et al. (2014) Contradictions in X-ray structures of intermediates in the photocycle of photoactive yellow protein. Nat Chem 6:258-9|
|Schotte, Friedrich; Cho, Hyun Sun; Kaila, Ville R I et al. (2012) Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc Natl Acad Sci U S A 109:19256-61|