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. We initiated another signifiant upgrade of the BioCARS beamline in 2014-15, in which a secondary K-B mirror pair and a new x-ray diffractometer was integrated into the end station. This secondary mirror system allows us to significantly reduce and control the x-ray spot size at the sample location. Time-resolved Laue diffraction images are acquired 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 can 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 protein's 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. The experimental capabilities developed on the BioCARS 14-IDB beamline at the Advanced Photon Source were exploited to track the reversible photocycle of photoactive yellow protein (PYP) following trans to cis photoisomerization of its p-coumaric acid (pCA) chromophore over ten decades of time. This protein is an excellent model system for probing the time-ordered sequence of events that lead to a signaling state, and allowed us to explore at a near-atomic level of detail how signaling proteins function. 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 novel structure is short lived (600 ps), has not been observed in prior cryo-crystallography 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. 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.