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Structure of a Protein Photocycle Intermediate by Millisecond Time-Resolved Crystallography

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Science  07 Mar 1997:
Vol. 275, Issue 5305, pp. 1471-1475
DOI: 10.1126/science.275.5305.1471

Abstract

The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.

Photoreceptors link light to life. Yet, understanding the molecular mechanisms for light-induced signal transduction has been limited by difficulties in obtaining and stabilizing light-activated conformations of suitable protein samples long enough for conventional structural studies by nuclear magnetic resonance or x-ray diffraction. Thus, three-dimensional structures known for photoactive proteins (1, 2) all describe proteins in their dark-state conformations. Here we present the structure of the light-activated, long-lived intermediate (I2) in the photocycle of PYP, as determined by time-resolved, multiwavelength Laue x-ray diffraction at a spatial resolution of 1.9 Å and a time resolution of 10 ms. This structure is expected to be the biologically important signaling state.

PYP is the 125-residue, 14-kD cytosolic photoreceptor (3, 4) proposed to mediate negative phototaxis (5) in the phototrophic bacterium Ectothiorhodospira halophila. The photocycle kinetics in PYP crystals (6, 7) resemble those in solution (4, 8). After photon absorption (wavelength of maximum absorbance λmax ∼446 nm), ground-state PYP (P) converts rapidly (≪10 ns) to a red-shifted intermediate (I1), then quickly (k ≈ 1 × 104 s−1) to a bleached, blue-shifted intermediate (I2). Spontaneous return of I2 to P by a relatively slow process (k ≈ 2 to 3 s−1) completes the photocycle. One proton is taken up by PYP during formation of I2 and released upon return to P (9). The 4-hydroxycinnamyl chromophore (Fig. 1A), covalently attached to Cys69 through a thioester linkage, is proposed to photoisomerize during the photocycle (10, 11). In the ground- or dark-state structure of PYP determined at 1.4 Å resolution (2), the yellow, anionic chromophore (10, 12) forms a hydrogen bond with a buried glutamic acid within a hydrophobic core, protected from solvent.

Fig. 1.

Chromophore structure (left) and difference (|Fphotostationary| − |Fdark|) electron density map with PYP fold in the ground state (white ribbon) and trans-chromophore (yellow) (right). The density map (contoured at 3σ) shows an excellent signal-to-noise ratio for the transient, light-induced, structural changes. The largest signal is localized at the active site (blue, positive; and red, negative electron density). Figures 1 to 4 were made with AVS (28).

The short lifetime of the I2 intermediate and the need to simultaneously record optical data presented challenges beyond those encountered in previous Laue crystallographic studies (13). Specific features of our experimental system and techniques contributed to the success of this study (14). PYP crystals diffract strongly, have low mosaic spread, allow repeated laser-triggering of the photocycle, and are relatively resistant to radiation damage. However, to avoid degradation of crystalline order and interference with optical measurements during continuous laser illumination, we collected data with the laser off during the decay from a saturated photostationary state established by off-peak laser illumination (14). The exciting laser, microspectrophotometer (15), and x-ray shutters (16) were synchronized (7, 17) for coordinated optical and diffraction data collection, and multiple exposures increased diffraction intensities (14).

Structural changes in PYP after light activation were localized near the chromophore (Fig. 1) in difference electron density maps produced from independent diffraction data processing by two methods (18). The data set collected 2 to 12 ms after laser shut off and processed with LaueView (19) provided optimal merging and wavelength scaling statistics (Table 1). Both high resolution and deconvolution of energy overlaps contributed to map quality, as assessed by comparison of electron density maps.

Table 1.

Laue x-ray diffraction data. Cum., cumulative.

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During the 2- to 12-ms time point in the decay from the photostationary state, ∼50% of PYP molecules exhibited significant active-site structural differences from the ground state (Fig. 2), including isomerization of the chromophore. This photostationary-state structure was refined independently by conventional all-atom crystallographic refinement and by selected-atom refinement with extrapolated structure factor amplitudes (20). In all-atom refinement (20), a model for the bleached intermediate was fitted to difference (Fig. 2, A and B) and omit (Fig. 2, C and D) electron density maps, then dual conformations were refined by positional and occupancy refinement (Table 2). The resulting ∼50% occupancy of the bleached conformer is about half of that predicted from a simple kinetic model (4, 7, 8), suggesting that photocycle physics (for example, back reactions) precluded a homogeneous population of bleached molecules under our experimental conditions. Alternatively, optical bleaching might not be directly coupled to structural changes observable by x-ray diffraction (7). In selected-atom refinement (20), 50% occupancies were used to extrapolate structure factor amplitudes for a hypothetical fully bleached crystal, and the bleached conformer was refined (Fig. 2, E and F). These diffraction amplitudes were in turn used for simulated annealing refinement (Table 2) in which only active-site residues associated with peaks in electron density difference maps (Fig. 2, A and B) were allowed to move. These two refinements of the bleached conformer of PYP showed light-induced structural changes that were identical within experimental error.

Fig. 2.

Atomic positions for the bleached (white) and dark (yellow) states of PYP's active site with the three different electron density maps used for structure determination. All three show a light-induced increase in the population of the bleached state. (Top panels) Arg52 is shown above the phenolic ring of the chromophore. (Bottom panels) The chromophore is shown beneath Arg52 (left) and Tyr42 and Glu46 (above). (A and B) Difference map (|Fphotostationary| − |Fdark|) (contoured at 2σ). Blue contours depict electron density that appears in the photostationary state; red contours depict diminished electron density. The ball of electron density (red) near Arg52 (A) is due to the movement of a water molecule (not shown) upon photobleaching. (C and D) Simulated-annealing omit map (|Fphotostationary| − |Fcalculated|) calculated for a model in which Arg52 and the chromophore were omitted (contoured at 1σ). The ratio of dark-state and bleached-state structures in the experimentally achieved photostationary state is approximately 1 to 1. (E and F) Extrapolated, simulated-annealing omit map corresponding to 100% population of the bleached structure (20) calculated with phases from the dark-state model in which Arg52 and the chromophore were omitted (contoured at 1.5σ).

Table 2.

Crystallographic refinement of photostationary state structure. Refl., reflections; Compl. completeness; conf., conformations.

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In the bleached structure of PYP denoted I2, the 4-hydroxycinnamyl chromophore has undergone a light-induced trans-to-cis isomerization around the carbon-carbon double bond that is conjugated with, and located between, the phenolic ring and the thioester linkage to Cys69 (Fig. 3). In the photobleached cis-chromophore, collision of the thioester carbonyl with the nearest aromatic ring proton produces a strained nonplanar conformation (by ∼60°) that could provide the driving force for return to the dark-state trans-isomer. In the bleached structure, the chromophore's aromatic ring has moved toward the protein surface, so that its phenolic oxygen atom is centered in the dark-state position of the Arg52 guanidinium group (Fig. 2, B and D). Arg52 has moved and reannealed to the protein surface in a new position. Consequently, the phenolic oxygen atom of the cis-chromophore becomes solvent-exposed and protonated, accounting for the proton uptake measured during formation of I2 (9). Residues 42 and 45 to 51, which were neighbors of the trans-chromophore, have moved inward to partially fill the cavity left behind by the movement of the chromophore. The hydrogen bonding network that stabilized the chromophore and Arg52 in the dark-state structure (2) has undergone major rearrangement. A single hydrogen bond to the phenolic oxygen of the chromophore from Arg52 (Fig. 3A) has replaced the two dark-state hydrogen bonds from Glu46 and Tyr42 (Fig. 3B). These changes affect the properties of the active-site surface (Fig. 4). Arg52 becomes more solvent-exposed (by ∼10 Å2) and forms only a single intramolecular hydrogen bond (Fig. 3A), leaving two side-chain hydrogen donors available for interactions with other molecules. In combination with chromophore protonation, these structural rearrangements produce a patch of positive electrostatic potential (Fig. 4C). These changes in surface shape, electrostatic potential, and chemical complementarity could alter interactions of PYP with an unknown second molecule to trigger a signal transduction cascade that ultimately reverses the flagellar motor to produce negative phototaxis.

Fig. 3.

Active-site hydrogen bonding networks for bleached (A) and dark (B) conformations. Oxygen (red), nitrogen (blue), and sulfur (yellow) atoms are shown as balls, and hydrogen bonds as turquoise tubes. During bleaching, dark-state hydrogen bonds from the Tyr42 and Glu46 side chains to the trans-chromophore's deprotonated phenolic oxygen (top), and from the Arg52 guanidinium group to the carbonyl oxygen atoms of Tyr98 and Thr50 (left), are broken. The protonated phenolic oxygen of the cis-chromophore now forms a hydrogen bond to Arg52 (left). Hydrogen bonds of the Cys69 main-chain NH with the chromophore's carbonyl group (bottom), and of the Thr50 side-chain OH with the Tyr42 side-chain OH and the Glu46 main-chain carbonyl oxygen (top), are conserved. The bleached-state conformation of Arg52 can also be fitted and refined in a flipped orientation within the same planar electron density (Fig. 2), but the location of the guanidinium group and the hydrogen bond with the cis-chromophore are conserved.

Fig. 4.

Solvent-accessible molecular surface of PYP in (A) the dark state and (C) bleached state color-coded for electrostatic potential as calculated by DelPhi (29) (deep red, <−4 kT; white, neutral; dark blue, >4 kT). Partial charges were assigned according to a revised version of the CHARMM force field (30). In (B), Cα traces and side chains of Arg52 for the dark (yellow) and bleached (white) states are shown. Bleaching increases the positive electrostatic potential at the active site. Movements of Arg52 and the chromophore change the surface shape.

In the bleached PYP structure, the protein remains well ordered, has undergone conformational rearrangements beyond those required to avoid interatomic collisions with the isomerized chromophore, and has formed a new set of active-site hydrogen bonds, distinct from those in the dark state. These structural features are characteristic of a protein at an energy minimum, rather than in a state of acute, steric perturbation. On the basis of the structure of I2 and photocycle kinetics of PYP (4, 7, 8), we propose a simple, structural model for the PYP photocycle. Photon absorption by the protein-bound chromophore transforms the dark or ground state (P) into the electronically excited state P* and rapidly leads to trans-to-cis isomerization of the chromophore to form the early intermediate I1. The extreme speed of the equivalent reaction in rhodopsin and bacteriorhodopsin (21) suggests that the P*-to-I1 transition in PYP is too fast to allow substantial rearrangement of the protein. Thus, the I1 structure would combine cis-chromophore geometry with a ground-state protein conformation. The chromophore isomerization would then trigger protein structural changes to achieve a new energy minimum, denoted I2. After cis-to-trans chromophore reisomerization driven by physical strain in the nonplanar cis-conformation, the protein will again rearrange to the dark-state energy minimum (P), completing the photocycle. Therefore, in our model, the PYP photocycle divides into two similar halves, each characterized by fast generation of new chromophore geometry followed by slower protein rearrangement to achieve a local energy minimum. The more complex photocycles of other light-activated proteins such as rhodopsin and bacteriorhodopsin can be described as extensions of this model with additional intermediates during the protein rearrangement steps. Thus, the bleached structure and associated photocycle model for PYP provide not only the structure of a prototypical intermediate in protein-mediated signaling, but also an exemplary framework for understanding the structural mechanisms of protein photocycles.

Note added in proof: Further information about the function of Glu46 and Arg52 has recently been obtained by time-resolved spectroscopy on site-directed PYP mutants (31).

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