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

Abstract

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.

To watch a protein function in real time with atomic resolution remains an elusive goal of molecular biophysics. This ability will help remove the mystery regarding how proteins execute their targeted function with remarkable efficiency and extraordinary selectivity, and replace it with a rational, molecular-level description of their reaction pathways. In a step toward this goal, we have 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 a photoactive signaling protein over 10 decades of time, from 100 ps to 1 s.

Signaling proteins give cells the capacity to respond to environmental changes and are crucial to an organism’s survival. The messenger to which a cell responds can be chemical, thermal, electrical, or light. Regardless of the source, the messenger typically triggers conformational changes in a signaling protein, whose altered structure leads to a regulatory response in the cell. Although static crystal protein structures provide clues regarding how a signaling protein might accomplish its target function, a complete mechanistic understanding requires knowledge about the sequence of structural changes that lead to the signaling event. When the messenger is chemical, the time required to diffuse to and bind in the active site of a signaling protein is typically far longer than the timescale for protein conformational change. Therefore, chemical-based signaling is not readily amenable to structural studies on fast time-scales. In contrast, when the messenger is light, a laser pulse can be used to trigger a photoreaction at a well-defined instant of time, and the ensuing structural changes can be followed via time-resolved Laue crystallography (1), a pump-probe methodology first demonstrated with nanosecond time resolution (2) and later extended to the picosecond time domain (3).

Light-induced signaling is ubiquitous in nature and includes vision in higher animals, phototropism in plants, and phototaxis in bacteria. For this study, we focused our attention on photoactive yellow protein (PYP), a 14-kDa water-soluble blue-light receptor first discovered in Halorhodospira halophila (4), a purple sulfur bacterium that swims away from blue light and toward photosynthetically useful green light (5, 6). Because its action spectrum for negative phototaxis matches the absorbance spectrum of PYP (6), this protein is presumed responsible for the signal that causes this extreme halophile to swim away from photons energetic enough to be genetically harmful. Since its discovery, PYP has served as a useful model system for biophysical studies of signaling proteins (7). The chromophore in PYP is p-coumaric acid (pCA), which is covalently linked to the Cys69 residue via a thioester bond (Fig. 1; Movie S1). The C2=C3 double bond is trans in the ground state (pG), but upon absorbing a single photon of blue light 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 (8–11).

Fig. 1.

PYP structure and photocycle. (A) Surface rendering (gray) of PYP (PDB ID code 2ZOH) in the pG state with backbone structure (ribbon) and atomic rendering of pCA and its hydrogen-bonding partners. Arrows point to the C2=C3 double bond (pCA) and the Cα atoms of key residues. Hydrogen bonds are indicated with dashed blue lines. Arg52 is stabilized in its “closed” state via hydrogen bonds to the protein backbone. [rendered with VMD (www.ks.uiuc.edu/Research/vmd/)] (B) The C2=C3 double bond in pCA is trans in its ground state; it absorbs blue light and gives PYP its yellow color. Photoisomerization from trans to cis triggers the PYP photocycle. (C) The PYP photocycle is labeled and color-coded according to intermediates characterized in this study. Intermediates colored gray and connected by dashed lines were not observed in this study, but have been characterized or implicated in prior studies.

Here, we present a comprehensive description of the structural changes occurring during the PYP photocycle. Based on the structures determined in this work, which are cross-validated with density functional theory (DFT) calculations, we propose a model for the PYP photocycle that is both chemically and mechanistically sensible, and accounts for much of the kinetic complexity observed in prior studies.

Results

Picosecond time-resolved snapshots of the PYP structure were acquired using the pump-probe method (3) (Fig. 2). Briefly, a laser pulse (pump) photoactivates a PYP 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 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. For this study, diffraction data from 41 orientations using 9 different crystals were combined to produce time-resolved electron density maps at 42 different time-points spanning 10 decades. To mitigate the adverse effects of radiation damage during data collection, very large crystals (12) were used (Fig. S1). Spreading the X-ray dose over the entire length of each crystal maintained high-quality diffraction throughout each of the 41 time-series. To improve the accuracy of the structural information contained in the 1.65-Å resolution Laue diffraction images, we developed an interpolated ratio method for processing the images (SI Materials and Methods).

Fig. 2.

Pump-probe geometry used to acquire time-resolved diffraction snapshots. The PYP crystal is sealed in a thin-walled glass capillary. Because the laser penetration depth in PYP is shallow, an orthogonal pump-probe geometry is used in which the top edge of the protein crystal is positioned at the top edge of the focused X-ray pulse. This geometry ensures optimal overlap between the laser and X-ray illuminated volumes of the crystal. The protein crystal acts as a monochromator with various line spacings (d) and diffracts different X-ray colors (λ) in different directions (θ) according to Bragg’s law (λ = 2d sinθ). Approximately 3,000 spots are found in each time-resolved diffraction image. The spots in this figure are annotated according to integrated photons (spot dimension) and X-ray wavelength (spot color).

The earliest structural event, the trans-to-cis photoisomerization of pCA, occurs in ∼1–3 ps (13–17) and is too fast to track with 150-ps time resolution. Nevertheless, the structural changes arising from this transition are captured in the 100-ps snapshot shown in Fig. 3 (Movie S2 depicts the structural changes observed throughout the time series). Although structural changes are evident throughout the protein, they are most dramatic in the vicinity of the pCA chromophore. Indeed, the refined pCA structure for the first intermediate detected, which we denote pR₀, is highly contorted with its carbonyl oriented ∼90° out of the plane of the phenolate (Fig. 3, curved arrows). This unusual structure has not been trapped in cryotrapping experiments (18–20), and its lifetime is too short to be captured in prior nanosecond time-resolved Laue crystallography experiments (21–23). The driving force for generating this intermediate is the trans-to-cis photoisomerization of pCA, during which the distance between the Cys69 sulfur and the terminal phenolate oxygen atom (Fig. 1B) contracts by ∼0.7 Å (Table S1). The pCA chromophore acts like a winch, pulling Glu46 and Tyr42 downward and Cys69 upward (Fig. 3, yellow arrows), thereby shrinking the corresponding Cα–Cα distances between these residues (Table S1) and generating strain in the protein. Remarkably, all three hydrogen bonds between pCA and the protein remain intact, but are clearly strained, as evidenced by longer hydrogen bonding distances to Glu46 and the Cys69 backbone (Table S1). The pR₀ intermediate sets the stage for the transitions that follow and ultimately lead to the long-lived PYP signaling state.

Fig. 3.

Front and side views of time-resolved structural changes recorded 100 ps after photoexcitation of PYP. (Lower) Expanded, annotated view of Upper Insets. The ground-state electron density map is colored magenta, and the 100-ps map is colored green. Where magenta and green overlap, the electron density blends to white. The magenta-to-green color gradient unveils the direction of atomic motion; large-amplitude displacements are indicated with yellow arrows. The stick models correspond to refined structures for the ground state (pG) and the first intermediate (pR₀). Hydrogen bonds to the pCA chromophore are indicated as dotted lines.

Later in the PYP photocycle, multiple intermediates are simultaneously populated. To determine their structures, we have developed a global data analysis methodology (SI Materials and Methods). Starting from a plausible kinetic model, we recover accurate structures of intermediates and accurate rate parameters for interconversion between them. Four intermediates are required to account for our experimental time-resolved electron density maps (Movie S2), the last of which is similar to a structure that has been assigned to a blue-shifted species (22, 24). Hence, we denote these four intermediates pR₀, pR₁, pR₂, and pB₀ (Fig. 4; Movies S3 and S4). Structures similar to pR₁, pR₂, and pB₀ have been reported previously (21, 22, 24, 25), but pR₀ is unique. The simplest possible kinetic model connecting these intermediates is 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 (Fig. 4A). This simple model accounts for the experimental electron density maps with high fidelity, as illustrated in the calculated vs. experimental color-coded overlays shown in Fig. S2. The ability to assess structurally the validity of a proposed kinetic model, as has been done here (SI Materials and Methods), is crucial to understand a protein’s mechanism, and is a capability unique to time-resolved crystallography.

Fig. 4.

Time-resolved population of transient intermediates and their structures. (A) Kinetic model used to account for structural changes spanning 10 decades; the arrows are labeled with the inverse of the globally refined rate constants. Half the population short-circuits to the ground state during the pR₂-to-pB₀ transition. (B) Time-dependent populations of each intermediate in the PYP photocycle: theoretical population predicted by the kinetic model (solid lines) and least-squares contributions from the four electron-density base maps (filled circles). (C–F) Structures for pR₀, pR₁, pR₂, and pB₀ intermediates. (Left and Center Left) Electron-density base maps were derived from global analysis and phased according to their refined structures (front and side views; see Fig. S3 for comparison with maps generated using ground-state phases). (Center Right) Refined structures of pCA intermediates and their hydrogen-bonding partners. To highlight the structural changes leading to the corresponding intermediate, they are overlaid with a semitransparent structure (gray) of the preceding state. (Right) Color-coded rendering of the protein backbone according to Cα displacement relative to pG, as indicated by the scale (rendered with VMD).

Discussion

The pR₀ structure unveiled here 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. This unusual conformation begs the question regarding how this ∼90° twist is accommodated among the three dihedral angles between the phenolate and the carbonyl; i.e., is pR₀ a strained cis intermediate with the ∼90° twist distributed among all three dihedral angles, or is the ∼90° twist confined to the C1′–C3=C2–C1 dihedral angle (see notation in Fig. 1B) and represents a transition state-like conformation between trans and cis? With the limited resolution of the crystallographic electron density, this question could not be answered unambiguously, because the refined structure depends on the dihedral angle restraints used in the refinement (SI Materials and Methods). Hence, we sought further guidance on this issue from DFT calculations (SI Materials and Methods). According to the DFT-optimized structure for pR₀, the C1′–C3=C2–C1 dihedral angle is displaced ∼30° (Table S1) from its classic cis conformation, and should therefore be considered a strained cis conformation. To accommodate the full ∼90° twist between the phenolate and the carbonyl, other dihedral angles are distorted as well. The X-ray–refined structure is consistent with the DFT-optimized structure provided the variance of the three pCA dihedral angle restraints used in the X-ray refinement are tightened from the default value of ±30° to ±4° (SI Materials and Methods). The X-ray–refined and DFT-optimized structures are found to be in excellent agreement for all other intermediates as well (Fig. S4), thereby cross-validating these structures and confirming the plausibility of our pR₀ structure.

Our characterization of pR₀ as a strained cis conformation is strongly supported by kinetic arguments as well. The equilibrium established between pR₀ and pR₁ precludes the possibility that pR₀ is a ∼90° twisted, transition state-like conformation, because the energy required to break the cis double bond in pR₁ and return to pR₀ would be far too high. Moreover, our finding that the quantum yield for producing pR₂ from pR₀ is near unity would be highly unlikely if pR₀ was a transition state-like species, from which return to the trans pG ground state should be equally likely. According to DFT energy calculations (SI Materials and Methods), pR₀ was found to be similar in energy to pR₁, but pR₂ is ∼7 kcal⋅mol⁻¹ lower (Table S1). These results are fully consistent with our observation that the transition between pR₀ and pR₁ is reversible, but the subsequent transition leading to pR₂ is irreversible.

The intermediate structures unveiled in this study permit a detailed examination of mechanistic issues in the PYP photocycle. To facilitate visual comparison of these intermediates, Fig. 4 shows their structures in three different views: color-coded electron density maps of pCA and its immediate surroundings; refined structures for pCA and relevant side chains; and color-coded renderings of protein backbone displacement.

The ultimate fate of pR₀ is dictated by the relative strength of the hydrogen bonds between the chromophore and protein. In wild-type PYP, the hydrogen bond between the Cys69 backbone nitrogen and the pCA carbonyl must be weaker than the phenolate hydrogen bond to Glu46, because the pR₀-to-pR₁ transition involves rupture of the hydrogen bond to Cys69. In the Glu46Gln mutant of PYP, the opposite would be true, and pR₀ (should it exist in this mutant as well) would be expected to transition to the first intermediate recovered (pR_E46Q) in prior nanosecond time-resolved Laue studies (26); that structure is similar to pB₀ except the phenolate is hydrogen bonded to Tyr42 instead of Arg52. To be consistent with the nomenclature presented here, we refer to that state as pR₃, because it is further along the pathway to pB₀ than is pR₂.

The pR₀-to-pR₁ transition enables the pCA to assume a planar configuration, which relieves some strain in the protein and the chromophore, but at the cost of breaking a hydrogen bond. According to DFT calculations, the hydrogen bond energy between the pCA carbonyl and the Cys69 backbone is 8.4 kcal⋅mol⁻¹ (Table S1). The pR₀-to-pR₁ transition is reversible, with the two populations quickly establishing an equilibrium that favors pR₁ ∼2:1. It follows that the magnitude of the strain energy relieved by the pR₀-to-pR₁ transition must be ∼9 kcal⋅mol⁻¹, ∼6 kcal⋅mol⁻¹ of which comes from the pCA chromophore alone (Table S1).

The pR₀-to-pR₁ transition leaves the C2–C1–S–Cβ dihedral angle in a sterically strained syn conformation (Table S1). The pR₁-to-pR₂ transition relieves this strain via a dihedral rotation to an anti conformation. According to DFT calculations, the strain relieved by the pR₁-to-pR₂ transition is approximately half that relieved by the pR₀-to-pR₁ transition.

The pR₂ state is the most stable red-shifted intermediate observed in this study, and eventually transitions to pB₀ with a time constant of 410 μs. Compared with the first two structural transitions, which involve localized motions, the pR₂-to-pB₀ structural transition is complex, and requires breaking two short (and strong) hydrogen bonds between the phenolate and Tyr42/Glu46, breaking two hydrogen bonds between Arg52 and the protein backbone, and ∼180° rotation of one of the pCA dihedral angles. The transition state for this complex reaction pathway likely imposes significant strain on the C2=C3 bond, which would account for our finding that approximately half the pR₂ population short-circuits to the ground state during this transition.

During the pR₂-to-pB₀ transition, Arg52 switches to an “open” conformation that exposes the pCA phenolate to water (Fig. S5) and facilitates its protonation, an event that triggers a blue shift of the pCA absorbance spectrum (27). Indeed, the pB₀ intermediate has a water molecule hydrogen bonded to the phenolate and the protein backbone. The open Arg52 conformation also facilitates penetration of a water molecule into the pCA cavity, which is stabilized by hydrogen bonds to Tyr42 and Glu46. Because this water molecule must escape before the pB₀ state can revert to the pG state, its presence likely prolongs the lifetime of the pB₀ state. Note that the pB₀ state reported here does not have the same global structure as that produced in solution, where the N-terminal domain is free to move beyond the constraints imposed by crystal packing. In our kinetic model, the crystal-inaccessible “signaling” intermediate, in which the N-terminal domain is believed to be partially unfolded (28–30), is denoted pB₁.

Changes in backbone displacement during the PYP photocycle are clearly evident in the color-coded (blue-white-red) ribbon structures of Fig. 4. These maps show a diminution of red coloration as the photocycle advances from pR₀ to pR₁ to pR₂, indicating relaxation of protein strain. The pR₂-to-pB₀ transition, however, exhibits large amplitude, globally distributed displacements of the protein backbone. Although the putative pB₁ signaling state is not achieved in the protein crystal, substantial motion in the N-terminal domain is detected during the pR₂-to-pB₀ transition, which is consistent with this region being implicated in the formation of the signaling state.

According to DFT energy calculations (SI Materials and Methods), the earliest intermediates store approximately one-third of the photon energy in the form of protein strain, which provides ample excess energy to drive the photocycle to the signaling state and back down to the ground state. The driving force for the final pB₀-to-pG transition should be sufficiently high to avoid thermal activation of pB₀. This driving force appears to reside primarily in intramolecular strain in the pCA chromophore and the relative strength of its hydrogen-bonding interactions. The pB₀-to-pG transition not only relieves the strain imposed by the cis conformation of pCA, but also exchanges two weaker hydrogen bonds for two short, strong hydrogen bonds. Due to the energetic cost of reversing this transformation, thermal activation of the pG state is an improbable event, as desired for a photoactive protein.

The lifetime of a signaling state must be sufficiently long for an organism to recognize and act upon its message, but not too long to remain stuck in the “on” state. H. halophila responds to changes in blue-light intensity in ∼0.5 s (6), which is of the same order of magnitude as the 0.26-s pB₀ lifetime reported here. This lifetime is determined by the height of the activation barrier for ground-state recovery, which is likely dominated by the energy required to break the C2=C3 cis bond. Thus, the pCA prosthetic group, which is capable of trans-to-cis photoisomerization, provides an energetically and structurally attractive framework for inducing and sustaining a long-lived signaling state in PYP.

The PYP photocycle found here differs from what was reported in earlier nanosecond time-resolved Laue crystallography studies (22, 23). Those studies lacked the time resolution to capture pR₀, but managed to characterize pR₁, which was believed to bifurcate to both pR₂ and pR₃ with pR₂ favored 3:2. If pR₃ is present in this study, its population is too small to be characterized. Moreover, those studies did not report water penetration into the pCA cavity (22, 23), except at pH 4 (23), and found the ground-state recovery from pB to be biexponential, with most of the population converting to pG on a 10- to 20-ms time scale (22, 23). Here, the ground-state recovery from pB₀ is exponential with a much longer, 260-ms time constant. The differences between this and earlier studies cannot be ascribed solely to improvements in data quality and the data analysis procedure, but appear to be due to differences in sample preparation. Extreme halophiles tolerate a high intracellular NaCl concentration. For example, Halobacterium salinarum grown in 4 M NaCl has been shown to have an intracellular NaCl concentration of 3.6 M (31). Nevertheless, most studies of PYP have been performed under conditions with relatively little or no NaCl present. In contrast, this study was carried out with P6₃ PYP crystals grown with 1.1 M NaCl in D₂O, pD 9.0. Under these conditions, which are arguably more physiologically relevant compared with prior crystallography studies, the PYP photocycle can be described by a simple, sequential model with four well-defined structural intermediates, whose lifetimes correlate favorably with those measured in solution studies. Similar results have been obtained at pH 7.0, demonstrating that the differences between this and earlier work are not due to pH or hydrogen/deuterium isotope differences. Evidently, the presence of 1.1 M NaCl mediates penetration of water into the pCA cavity, which accounts for the long-lived pB₀ state reported here. Interestingly, the longer-lived pB population reported in earlier studies (22, 23) has a lifetime similar to what we find here, and may arise from water occupancy in the pCA cavity that is too low to be detected in the maps.

The intermediates found in this work provide a structural framework for interpreting the wealth of prior time-resolved spectroscopic studies of PYP in solution, a few examples of which are presented here. (i) Transient optical absorbance spectra of PYP in solution (13, 14) unveiled a short-lived intermediate peaked at ∼510 nm that relaxed to a longer-lived feature peaked at ∼460 nm. We associate the short-lived 510-nm intermediate with pR₀ and the longer-lived 460-nm intermediate with pR₁ and pR₂, which are similar in structure and would be expected to have similar optical absorbance spectra. The lifetimes reported for the short-lived intermediate, 3.0 ns for I₀^‡ (13) and 1.3 ns for PYP_B (14), are longer than the 600-ps lifetime we find for pR₀; however, the lifetime recovered in this study is expected to be shortened by the unavoidable <30 K adiabatic temperature jump that arises when photoactivating PYP crystals with the laser pulses used in this study. (ii) Prior studies uncovered an equilibrium between red- and blue-shifted intermediates (32, 33). We find no evidence for an equilibrium between pR₂ and pB₀, and would not expect it for reasons already discussed; however, if pR₃ were formed in solution, it would be expected to coexist in equilibrium with pB₀. A mixture of pR₂ and pR₃ would also account for the biexponential kinetics reported for the conversion of pR to pB (8). (iii) Hendriks et al. (32) characterized the PYP photocycle over a wide range of pH and pD, and in table 1 of ref. 30 reported relaxation times at pH 8.1 and pH 9.55, whose averages are remarkably similar to what we find in PYP crystals at pD 9.0: 420 (410) μs for pR to pB, and 220 (260) ms for pB to pG (our determinations in parentheses). The fact that the pR and pB lifetimes in crystals grown in ∼1.1 M NaCl are similar to those found in solution at a similar pH/pD (32) suggests that the pCA photocycle in our crystals is authentic, and is only loosely coupled to the large-amplitude displacement that accompanies the putative pB₀-to-pB₁ signaling transition.

The time-resolved structural evolution presented here, spanning 10 decades, unveil a simple, step-wise structural progression of PYP conformations toward a long-lived pB₀ state, with each transition characterized at an unprecedented level of detail. The highly contorted structure of the pR₀ state is unique, and provides a visual clue regarding the conformational gymnastics that must accompany trans-to-cis isomerization in a highly constrained protein environment. These results illustrate how a protein can use hydrogen bonding networks, gated water penetration, and strain to steer the direction of structural transitions in a fashion that facilitates its target function. The time-resolved methodology developed for this study of PYP is, in principle, applicable to any other crystallizable protein whose function can be directly or indirectly triggered with a pulse of light. Indeed, it may prove possible to extend this capability to the study of enzymes, and literally watch an enzyme as it functions in real time with near-atomic spatial resolution. By capturing the structure and temporal evolution of key reaction intermediates, picosecond time-resolved Laue crystallography can provide 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.

Materials and Methods

Data reduction and analysis methodologies are described in SI Materials and Methods, which includes Figs. S6–S10. Data collection statistics are found in Table S2, and refinement statistics are found in Table S3.

Crystal Preparation.

Wild-type PYP apoprotein was overexpressed in Escherichia coli, isolated, and then reconstituted with pCA anhydride in 4 M urea buffer (34). The holoprotein was purified by column chromatography (DEAE Sepharose CL6B; Amersham Biosciences) several times until the optical purity index (absorbance 277 nm/absorbance λ_max) became <0.44. Large P6₃ crystals of PYP were grown from microseeds via the hanging-drop vapor diffusion method (12). The crystallization hanging-drop solution contained 24 mg⋅mL⁻¹ PYP, 2.2 M ammonium sulfate, 1.0 M sodium chloride, and 20 mM sodium phosphate. The reservoir solution contained 2.5 M ammonium sulfate and 1.1–1.2 M sodium chloride, and was maintained at 293 K. The crystallization buffers were prepared with 99.9% heavy water (Aldrich) and titrated to pD 9.0. This method produced high-quality crystals ∼2 mm in length. The crystals were mounted in 1.5-mm diameter glass capillaries whose inner wall was texturized by bonding thin shards of crushed glass to its surface with polyvinyl formyl. Crystals mounted on the roughened surface are far less prone to slippage during data collection, which can be problematic due to the shock wave generated in the crystal after an intense, short duration laser pulse is absorbed and suddenly heats its surface.

X-Ray Source.

The data reported here were acquired on the BioCARS 14-IDB beamline (35) at the Advanced Photon Source in March 2011 when the ring was operated in hybrid mode. During this run, 12-keV polychromatic (5% FWHM), 120-ps–duration X-ray pulses with nearly 3 × 10¹⁰ photons were focused to an 80 × 40 (horizontal by vertical) μm² spot at the sample position.

Laser Source.

The picosecond laser system used in this study employs a Spectra Physics Spitfire Pro designed to produce 5 mJ 1.2-ps optical pulses at 780 nm. To produce ∼120-ps pulses, we used a 0.8-nm band-pass interference filter to bandwidth limit the seed pulses delivered to the amplifier pulse stretcher, and then bypassed the grating pulse compressor by inserting a large right-angle prism into the optical layout of the Spitfire Pro. The laser pulse was frequency doubled to 390 nm in a type I β-barium borate nonlinear crystal custom-designed for these pulse characteristics. The 390-nm laser pulses were beam expanded and transported from the laser hutch to the X-ray hutch where beam-conditioning optics spatially filtered the beam and focused it to a 0.12 × 0.6 mm elliptical spot with the long axis aligned along the propagation direction of the X-ray beam. The beam conditioning optics arrangement includes a variable attenuator to control the pulse energy and a Berek polarization compensator to set the laser polarization.

PYP Photoactivation.

The peak absorbance of the pCA chromophore in P6₃ crystals (447 nm) is very strong and birefringent (71.4/407.1 mm⁻¹ ||/⊥ to the c-axis) (36). To match the laser penetration depth to the 40-μm vertical X-ray beam size, the laser must be tuned well to the red or blue edge of the ground-state absorbance. Because the first intermediate in the PYP photocycle has a red-shifted absorbance spectrum, it is preferable to photoactivate PYP on the blue edge. Moreover, it is preferable to photoactivate the crystal with laser pulses polarized parallel to the c-axis, which maximizes the laser penetration depth and reduces the strong depth-dependent gradient that would arise from using either circularly polarized or unpolarized laser pulses.

The laser system and beam conditioning optics were set up to deliver intense (3.5 mJ⋅mm⁻²), polarized (|| to c-axis), stretched (125-ps) optical pulses tuned to the blue edge of the ground-state absorption spectrum (390 nm). Under these conditions, the laser penetration depth is only 28 μm (1/e). Use of a stretched, intense optical pulse to photoactivate the PYP crystal boosts the trans-to-cis isomerization yield by providing the pCA chromophore multiple opportunities to isomerize during the optical pulse.

To ensure optimal overlap between the laser and X-ray pulses, it is crucial to align the top edge of the crystal with the top edge of the X-ray pulse (Fig. 2). To accomplish this feat, we developed an edge-finder routine that scans the crystal vertically through an attenuated X-ray beam while acquiring single-shot diffraction images. The extrapolated edge of the crystal is deduced from the integrated spot intensities, and the crystal is positioned to align its top edge with that of the X-ray beam. With this approach, the photoisomerization yield within the volume probed by X-rays was ∼10%.

During data collection, the crystals were chilled to 288 K with a nitrogen-gas cooling stream. The energy deposited in the protein crystal by the laser pulse triggers an adiabatic temperature jump that is largest at the protein surface and decreases as a function of depth. Given the conditions used in this study, the magnitude of the temperature jump decreases from ∼30 K to 7 K across the 40-μm vertical cross-section of the X-ray beam. Because the thermal conductivity in a protein crystal is quite high and its heat capacity large, the temperature in the X-ray–illuminated volume of the crystal rapidly cools to the cooling-stream temperature. Consequently, the early-time dynamics are recorded at an elevated temperature, which would be expected to shorten the lifetimes of the earliest detected intermediates, whereas the long-time dynamics are recorded at a near-normal temperature. In contrast, the protein concentration in time-resolved optical spectroscopy experiments is comparatively low, so the adiabatic temperature jump in those studies is negligible.

Data Collection Protocol.

Time-resolved diffraction images were acquired at 42 time delays spanning 10 decades, with four time points per decade. Interleaved between these time delays were twelve −1-ns reference images in which the X-ray pulse arrived 1 ns ahead of the laser pulse. A complete time series was acquired at each of six or more orientations per crystal on nine crystals. To improve the signal-to-noise ratio of each diffraction image, five consecutive pump-probe shots were integrated on the mar165 CCD detector before readout. The crystal was translated ∼250 μm along its long axis after each laser shot to avoid buildup of long-lived states. The time required for crystal translation and detector readout averaged 13 s per image. The starting position for each time point in the series was offset horizontally to uniformly distribute the radiation dose over the entire length of the protein crystal. This approach minimized the adverse effects of radiation damage and preserved high-quality diffraction throughout the entire time series.