Structural waters define a functional channel mediating activation of the GPCR, rhodopsin

Abstract

Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H₂O¹⁸ solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.

Membrane embedded proteins evolved to facilitate water or ion movements both in signaling processes and ion transport across the biological membrane. Many multispanning transmembrane proteins contain hydrophobic cores with strategically placed ionizable or charged residues that appear critical for their proper function (1). Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules (2–10) that may act like prosthetic groups with activities distinct from bulk water. Identification of “ordered” waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus the observed waters likely represent a subset of the ensemble of tightly bound and functional waters (11). Overall, it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions.

Radiolytic footprinting of macromolecules in dilute aqueous solution was developed to analyze the solvent accessibility of macromolecular sites in nucleic acids and proteins as a function of macromolecule conformation, and a detailed understanding of the approach has been developed where radiolysis generates radicals in bulk solution that freely diffuses to macromolecular sites and covalently labels those sites (12–14). In proteins, the chemistry of modification is well-established, and many types of side chain can be efficiently labeled and the products easily detected by mass spectrometry, although the dynamic range of labeling varies considerably, with aromatic and sulfur containing side chains easily labeled and aliphatic and polar residues labeled less efficiently (15, 16). For soluble proteins, it is well-established that this intrinsic reactivity and the solvent accessibility of the side chains govern their observed reactivity (15, 17, 18). For membrane proteins, these approaches have not been previously explored, such that it was unclear what factors would govern labeling, or whether the overall scavenging effects of detergents or lipids are limiting.

For family A GPCRs, understanding the structure and activation of rhodopsin with its covalently bound ligand, 11-cis-retinylidene, has been greatly facilitated by obtaining X-ray crystal structures of vertebrate and invertebrate rhodopsins (10, 19), photo-intermediates with covalently bound all-trans-retinylidene (20–22), and inactive ligand-free end-product opsin (23, 24). For example, crystallographic studies of photoactivated Meta II-like rhodopsin reveal local structural reorganizations without large-scale conformational changes of individual domains of this receptor (22). Recent structural studies of other family A GPCRs, including β₁ and β₂ adrenergic and A_2A adenosine receptors, show conserved membrane topologies within this receptor family, indicating the potential for similar if not conserved activation mechanisms (2–5). A striking common feature of these receptor structures is the presence of conserved, crystallographically ordered clusters of water molecules located in the transmembrane core that make contacts with highly conserved residues in family A GPCRs (11). Water is known to be critical for the activity of rhodopsin, specifically to achieve the Meta II state (25), and water is also required for the formation of apo-protein opsin via hydrolysis of the chromophore, all-trans-retinal (26). Although, the importance of water has been demonstrated in another 7-transmembrane protein, bacteriorhodopsin (27), the molecular details of agonist-induced structural transformations, the changes accompanying chromophore hydrolysis in the signaling process, and the potential role for ordered waters in mediating signaling, are not well understood (28).

We used radiolytic protein footprinting to investigate the conformational dynamics of ground state (rhodopsin), photoactivated (Meta II), and inactive ligand-free receptor (opsin) in n-dodecyl-β-maltoside preparations and native membranes. Unlike our previous data on soluble proteins, radiolytic modifications were observed for residues located in both solvent-accessible and solvent-inaccessible regions; in the latter case, residues within the transmembrane domain were labeled, and their reactivity varied as a function of rhodopsin activation state. Based on H₂O¹⁸ exchange studies combined with radiolysis, we determined that labeling within the transmembrane region is partly derived from tightly bound waters and that changes in labeling reveal regions undergoing local structural alterations and water reorganization.

Results and Discussion

Radiolytic Labeling of Membrane Embedded Domains of Rhodopsin.

Purified rhodopsin was exposed to high flux X-rays for 1- to 10-ms time periods. Although the X-ray-irradiated samples showed some loss of chromophore absorbance at 500 nm (Fig. S1), this analysis was conducted well after the millisecond irradiation, and gel analysis did not show a significant amount of amide bond cleavage (Fig. S2). Following exposure to high flux ionizing radiation, protein samples were digested and the resulting peptides were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS) (Fig. S3A). The extent of modification was quantified following extraction of ion current data for unmodified and modified peptides of interest. Typical mass spectrometric sequence coverage of the proteolytic fragments was 88% (Fig. 1A). Peptides not detected included sequences in the solvent exposed N-terminal extracellular region, which has been found not to undergo structural reorganization following photoactivation in crystallographic studies (22), and sequences in the extended cytoplasmic loop III (C-III), which are highly disordered in crystal structures (22), and a portion of helix VI. The remaining regions of rhodopsin were detected, in some cases with overlapping peptides. Rhodopsin exposed to X-rays either in native disk membranes or detergent exhibited roughly comparable extents of radiolytic labeling, and the labeling was on the same residues (Fig. S3 B and C). Identities of peptides and sites of modification were confirmed by tandem mass spectrometry (Fig. S3 D and E). Rates of modification were determined from X-ray dosage plots as described (Fig. S3F) (15, 16). The positions of the modified residues are shown in Fig. 1B; surprisingly labeled residues are observed throughout the transmembrane segment.

Fig. 1.

High resolution LC-MS analysis of proteolytic fragments of rhodopsin results in ≈88% amino acid sequence coverage. (A) The primary sequence of rhodopsin with proteolytic peptides detected and confirmed by tandem MS analysis is indicated by bars below the amino acid sequence. Peptides detected following digestion with pepsin are underlined by dark gray bars, and those detected after cyanogen bromide digestion and LC-MS analysis are underlined by light gray bars. Residues that make up transmembrane domains of rhodopsin are colored red. (B) LC-MS/MS analysis reveals radiolytic modification of many residues (yellow sticks) found in the membrane embedded region of rhodopsin as shown in this crystallographic model. The shaded rectangle represents the membrane; regions detected by LC-MS/MS analysis are colored green and undetected regions are colored gray. The chromophore, 11-cis-retinylidene, is shown as orange sticks. Ordered waters are shown as red spheres in the transmembrane domain.

Isotopic Labeling Reveals Limited Exchange of Bulk Solvent with Bound Waters Located in the Helical Bundle of Rhodopsin.

We used rapid mixing combined with O¹⁸-mediated hydroxyl radical labeling to determine the potential origins of the transmembrane labeling and to monitor the extent of exchange of bulk solvent into the core of the helical bundle. For most side chains, although the radiolytic labeling under aerobic conditions is mediated by hydroxyl radical, the oxygen atoms are derived from molecular oxygen. The notable exceptions are Phe, Tyr, Cys, and Met, where O¹⁸-labeling from radiolysis of H₂O¹⁸ is entirely derived from radiolysis of water (12, 16). As many of the labeled residues in the core are Phe, Cys, Met, or Tyr (Fig. 1B), we expected to detect O¹⁸-labeling for regions within the core of the receptor that experience increased bulk solvent exposure as a result of an opening of the helical bundle. Rhodopsin, Meta II, and opsin were mixed with H₂O¹⁸ containing buffer (in 1:1 ratio) just before X-ray exposure; in these experiments, there was no labeling from bulk solvent for either of the three receptor states except for the side chain of solvent accessible F13. Trace levels of O¹⁸-labeled Y306, Y301, and M86 were detected only following dehydration and rehydration with H₂O¹⁸-containing buffers (Fig. 2). Thus, the labeling of these residues at least is derived from tightly bound waters, including the ordered waters found in rhodopsin crystals. However, the fact that only a few internal residues are labeled with H₂O¹⁸ suggests that much of the transmembrane labeling is derived from a combination of direct radiolysis of the protein and radiolysis of nonexchangeable waters; the numbers of the latter may be substantial as indicated by the fact that 10% of the weight of a dried protein is because of tightly bound water (29). No labeling from bulk solvent is observed, and major structural rearrangements that would allow influx of bulk solvent during the time scale of photoactivation appears unlikely. These results are consistent with recent EPR measurements, which suggested that helical bundle reorganization upon activation is on the order of 6 Å or less, which in our opinion is not consistent with significant solvent exchange (30).

Fig. 2.

The extent of bulk water exchange into the membrane embedded domain of rhodopsin was measured by monitoring the incorporation of O¹⁸ from isotopically labeled hydroxyl radicals. Taking advantage of locations sensitive to hydroxyl radical oxidation, we monitored solvent uptake for several activated states of this receptor over time. Rapid mixing of H₂O¹⁸ containing buffer with rhodopsin, Meta II, and opsin was followed by equilibration (delay) periods of 50 ms, 500 ms, 5 s, and 30 s before X-ray exposure for 6 or 40 ms. No O¹⁸ hydroxyl radical incorporation occurred in any of these experiments except for the exposed amino terminal peptide. This indicates that radicals that modify residues within the helical bundle are formed in situ in intramembranous regions of rhodopsin, photoactivated receptor, and apo-protein. Only following dehydration and rehydration of rhodopsin with 97% H₂O¹⁸ water and subsequent X-ray exposure were low levels of O¹⁸ incorporation detected in resulting proteolytic fragments (A, blue ribbons) from the transmembrane domain (A, yellow spheres). For example, O¹⁸ labeling of M86 in peptide ⁸⁵FMVFGGF⁹¹ located in helix II was specifically detected by tandem mass spectrometry. The daughter ion spectrum of unmodified peptide ⁸⁵FMVFGGF⁹¹ is shown in panel B and the tandem MS spectrum of O¹⁶-modified M86 in panel C. Importantly, O¹⁸ hydroxyl labeling of M86 following X-ray exposure was only detected following full exchange of solvent after sample dehydration and rehydration with H₂O¹⁸ (D).

Water Functions as an Allosteric Modulator in Rhodopsin Activation.

In rhodopsin, ordered waters are within hydrogen bonding distance of highly conserved and functionally important residues such as D83, E113, W265, and the NPxxY motif (28). Inspection of several high-resolution crystal structures of family A GPCRs reveals a set of conserved waters, and those present in rhodopsin are shown in Fig. 1B (11). Interactions mediated by these ordered waters suggest that they are likely to play an important role in stabilizing helices I, II, III, and VII. The observed rate constants of radiolytic modification for residues in rhodopsin summarized in Table 1 do not show a precise correlation with the proximity to ordered waters revealed by crystallography. We suspect that there are “unobserved” waters that are relevant to the radiolysis as well and that the observed rate constants of modification reflect the nature and location of generated radicals, both protein and water, the mobility of hydroxyl radicals or other generated radicals, the local structure of the side chains, and the intrinsic reactivity of the residue. With these caveats in mind, comparative analysis of radical modification rate constants (only those >0.1 s⁻¹ are considered) for residues present in transmembrane helices II, III, IV, and VII of rhodopsin indicate structural changes near the chromophore-binding pocket and increased labeling of side chains in these helices following receptor activation.

Table 1.

Hydroxyl radical modification rate constants for residues in rhodopsin^†

Peptide‡	Rhodopsin (s−1)	Meta II (s−1)	Opsin (s−1)	Ratios of rate constants
				Meta II/ rhodopsin	Meta II/opsin	Opsin/rhodopsin
85FM*VFGGF91	7.4 ± 0.2§	22.6 ± 1.4	2.1 ± 0.4	3	10.8	0.3
113EGFF*116	238.1 ± 21	370 ± 16.5	254 ± 47	1.6	1.5	1
140C*KPM*SNF146	4.2¶ ± 0.5	8.0¶ ± 0.6	16.2¶ ± 0.9††	1.9	0.5	3.8
147RFGENHAI*M*G156	3.8¶ ± 0.4	6.6¶ ± 0.4	ND‡‡	1.7	–	–
160TWVM*A164	4.5 ± 0.5	13.6 ± 1.4	12.8 ± 1.5	3	1	2.8
176SRYIPEGM*Q184	1.8 ± 0.8	1.1 ± 0.5	1 ± 0.1	1	1.1	0.6
288M*TIPAF293	4.2 ± 0.3	7.6 ± 0.5	6.6 ± 0.4	1.8	1.2	1.6
293FAKTSAVY*NP*VIY*306	2.1¶ ± 0.5	1.1¶ ± 0.2	4.0¶ ± 1.1	0.5	0.3	2
333ASTTVSKTETSQV*A*P*A*348	8.1¶ ± 0.3	7.9¶ ± 0.4	6.8¶ ± 0.3	1	1	0.8

*, Denotes residue(s) modified by hydroxyl radicals.

^†Rate constants were estimated by employing a nonlinear fit of hydroxyl modification data to a pseudo first order decay as described in Materials and Methods (supplement).

^‡Peptides detected following LC-MS analysis and confirmed by MS/MS analysis with rate constants of modification suitable for quantitative analysis. As shown in Figure 1, other peptides exhibited modifications that were confirmed by tandem MS/MS.

^§Error in rate constant estimation calculated from nonlinear fit.

^¶Rate constants reflect mixed modifications.

^††Rate determined from peptide 137–146 with the same modified residues as peptide 140–146.

^‡‡Peptide not detected in this experiment, no rate is reported.

Release of Structural Constraint Resulting from Receptor Activation.

In Table 1, a comparison of rates constants of radiolytic labeling for selected peptides in ground and activated states (Meta II) of rhodopsin identifies regions of local conformational change associated with receptor activation. Following photoactivation of rhodopsin, a net loss of water upon Meta II formation has been described, and this may be linked to some of the dynamic changes seen here (31, 32). Additionally, rates of radiolytic modification for the apo-protein opsin provide insight into the structural dynamics specific to the deactivated state. These data reveal changes in regions known to undergo structural alterations following receptor activation. A 2-fold increase in radiolytic labeling was observed for C140 and M143 in helix III near the conserved ERY residues of the “ionic lock,” and increased sulfhydryl reactivity following photoactivation has previously been observed (33). Comparison of the rates of modification between Meta II and opsin reveals an even greater increase, suggesting the release of secondary structural constraints and dynamics of water and protein within Meta II following its transition to opsin.

Residue F116, located in helix III and in the plane of the chromophore, exhibited the highest rate of modification in all states of the receptor with modification rate constants ≈10-fold greater than those found for any other residue. We also note that this modification rate is much higher than we have ever previously observed for any Phe residue in a soluble protein. This residue is one turn down (C-terminal) on helix III from the Schiff base counter ion E113 (28), which was found to be within hydrogen bonding distance of crystallographically identified ordered water (atom number 2021 in 1U19.pdb). The very high rate of radiolytic modification of this site in all states of the receptor is possibly because of proximity of ordered water, and the increase in labeling upon activation may reflect movement of waters and/or side chains favoring the reaction. The reduced modification rate of F116 found in opsin may be because of disorder of residues near F116 upon receptor activation as seen in solid state NMR and crystallographic studies. Thus, in the opsin state, the water may not be positioned as favorably for reaction (22, 34, 35).

The largest relative change in the rate constants of radiolytic modification was observed for residue M86. This residue lies near a region of the receptor, namely residues 121–136, that exhibits an increased disorder following photoactivation (22, 34). We observed a 3-fold increase in the rate of radiolytic modification of M86 when comparing rate constants for ground state and activated receptor (Table 1). As for F116, this increase in modification was specific for the activated state, because the inactive state of the receptor (opsin) evidenced a 10-fold reduction in radiolytic modification as compared with Meta II. M86 is located in helix II, 3.9 Å (C-terminal) from the functionally critical and highly conserved residue D83. Interactions between helix II and VII are mediated by water via hydrogen bonding between D83, found in 94% of family A GPCRs, and residues S298, V300, and N302 located in helix VII, conserved in 75% of family A GPCRs (36). The functional importance of D83 has been demonstrated by a mutation that led to increased formation of Meta II (37, 38). These findings clearly demonstrate changes in conformational dynamics near the highly conserved and functionally important residue D83 differentiate the inverse agonist, agonist bound, and ligand-free states of rhodopsin.

Further away from the ligand-binding pocket toward the cytoplasmic face of the receptor, residue M163 in helix IV showed a 3-fold increase in modification of the activated versus the ground state of the receptor. The observed increased rate of modification of M163 was common to both Meta II and opsin. We interpret the increased conformational dynamics of M163 as the release of constraints mediated by the Y206-H211-M163-E122 hydrogen bond network holding helix III and V together in the ground state of the receptor, consistent with studies employing solid state NMR spectroscopy (35, 39). Residues ¹⁵⁴IM¹⁵⁵ exhibited a 2-fold increase in the rate of radiolytic labeling when ground state rates were compared with those of Meta II. This reflects the dynamics of specific residues at the cytoplasmic face of the receptor following release of the ionic lock holding helix III and helix VI together, likely reflecting movements of helix VI found to occur as a consequence of photoactivaiton (30). Residue M288, located in helix VII on the intradiscal side of the chromophore-binding pocket (28), was found in rhodopsin crystal structures near ordered waters (964 and 2014 in 1U19 in Protein Data Base). There was a 2-fold increase in the rate of radiolytic modification for this residue in the activated receptor and a subsequent modest decrease in modification rate for the inactive opsin. The observed increase in radiolytic labeling rate of M288 found for the activated state highlights local structural changes occurring near the ligand-binding pocket and indicate the presence of activated water. In contrast, the nearby residue M183 in the E-II loop, whose structure is indicated to be altered upon activation, did not show a similar increase in labeling rate, likely because of movement of water away from this site (35). These results demonstrate that radiolytic labeling of membrane proteins probes both local structural dynamics and hydration. A slight reduction in the rate of radiolytic modification was observed upon light activation for residues near the NPxxY domain in helix VII, with the rate for opsin being the highest of the three states; no state-dependent changes in modification rates were observed for the carboxyl terminal tail (Fig. 3 and Movie S1, Movie S2, and Movie S3).

Fig. 3.

Pictorial summary of modification rate constants. Radiolytic modification rate constants were determined for many residues in rhodopsin (Left), Meta II (Center), and opsin (Right). Residues with rate constants >0.1 s⁻¹ are rendered as spheres colored by rate constant ranges: 0.5–1.2 s⁻¹, light blue; 1.3–3.9 s⁻¹, light green; 4.0–5.9 s⁻¹, green; 6.0–7.9 s⁻¹, light-yellow; 8.0–9.9 s⁻¹, yellow; 10–14.9 s⁻¹, light-orange; 15–25 s⁻¹, orange; >200 s⁻¹, red. Following photoactivation, modification rates increased for M86, C140, M143, the pair of residues in helix IV I154 and M155, M163, and M288. Residues exhibiting decreased modification rates were Y301, P303, and Y306 in helix VII. There also was a reduced modification rate of M86 and F116 in opsin as compared with the two other states. The mixed modification of peptide 137–146, comprising part of the C-II loop, showed a large increase in the rates of detectable modification for opsin relative to ground state and activated rhodopsin, whereas M183 in the E-II loop exhibited no change in modification rate as a function of receptor activation state. The carboxyl terminal peptide did not show a marked difference in modification rates between the three states of the receptor. Changes in rates of oxidation observed when comparing ground state and activated receptor reflect local structural changes upon formation of both Meta II and opsin.

Conclusion

The above results suggest that the structural reorganization observed for Meta II does not simply stem from the release of water-mediated constraints that allow increased mobility of regions in helix II and helix VII, because the carboxyl terminal domain of helix VII exhibited a slight decrease in its observed rates of modification. Rather, we posit a directional increase in the activation-dependent rate of modification, highest near the chromophore-binding pocket and radiating toward the cytoplasmic face of the receptor along helix IV as evidenced by increased modification rates for residues M163 and ¹⁵⁴IM¹⁵⁵ (Fig. 3 andMovie S1, Movie S2, and Movie S3).

Upon light activation, the structural reorganization adjacent to retinal is obvious from many studies; in this work F116, M86, and M288 exhibited clear increases in labeling upon receptor activation (Fig. 3 and Movie S1, Movie S2, and Movie S3). However, this increased mobility did not extend to M183 or the conserved NPxxY motif as previously speculated (40). Instead, the suggested water reorganization extended to M163, M155, M143, and C140, and, by implication, to loop C-II. This may represent the linkage of the ionic lock on the cytoplasmic face of the receptor to a secondary internal hydrogen bonding network comprised of residues Y206-H211-M163-E122. Disruption of the secondary interhelical network, analogous to the ionic lock, may in fact be mediated by water reorganization, which explains in part the requirement for a hydrated receptor to attain the activated state. Our radiolytic footprinting of rhodopsin does demonstrate the structural activation of bound waters as a function of receptor signaling status and suggests a possible path of allosteric communication that may be conserved among family A GPCRs (36). For example, this pathway could be established by specific protonation/deprotonation of key residues such as E113 and E134 (28). Overall, we suggest a general framework where disruption and reorganization of multiple close packing interactions mediated by both side chains and bound water transmit sufficient information from the chromophore (ligand-binding site) to the cytoplasmic surface to promote catalytic exchange of GDP to GTP in the GPCR-bound G protein, transducin.

This work demonstrates the utility of radiolytic footprinting for the observation of structure and dynamics of the transmembrane region, including dynamics of water, in membrane proteins. This methodology is highly complementary to structural studies employing X-ray crystallographic, NMR, FTIR spectroscopy, and together, with these approaches, has the potential to define allosteric channels for other family A GPCRs, transmembrane signaling proteins, and ion channels.

Materials and Methods

Rhodopsin and Opsin Purification by 1D4 Immunoaffinity Chromatography.

Bovine rod outer segment (ROS) membranes were prepared from fresh retinas under dim red light (41). Soluble and membrane associated proteins were removed from these membranes by 5 washes with hypotonic buffer consisting of 5 mM sodium cacodylate, pH 6.5 or 7.2. Rhodopsin in n-dodecyl-β-D-maltoside was initially purified from ROS by the ZnCl₂-opsin precipitation method as previously described (42). Extracted rhodopsin was loaded onto a 1D4-coupled CNBr-activated Sepharose 4B column (binding capacity 0.5 mg protein/mL resin; Amersham Biosciences) equilibrated with Buffer A consisting of 10 mM sodium cacodylate, pH 6.5 (or 7.2), 100 mM NaCl, and 1 mM n-dodecyl-β-D-maltoside. The beads were washed with 10 volumes of the equilibration buffer and then 10 volumes of Buffer B consisting of 10 mM sodium cacodylate, pH 6.5 (or 7.2), and 0.4 mM n-dodecyl-β-D-maltoside. Purified rhodopsin was eluted with 100 μM TETSQVAPA, a nonapeptide from the rhodopsin C-terminal sequence, in Buffer B at room temperature. The concentration of purified rhodopsin was determined by measuring the absorption at 500 nm (ε = 40,600 M⁻¹ cm⁻¹) (43). For opsin purification, ROS membranes (1 mg/mL) resuspended in buffer consisting of 10 mM sodium cacodylate, pH 7.2, and 100 mM NaCl were illuminated in the presence of freshly neutralized 50 mM NH₂OH under the fiber light for 10 min at room temperature. Excess nucleophile then was removed with 4 washes of the above buffer. The resulting opsin was solubilized in 10 mM sodium cacodilate, pH 7.2, 100 mM NaCl, and 20 mM n-dodecyl-β-D-maltoside and purified as above. Opsin concentration was determined by Bradford ULTRA (Novexin) with BSA used as a standard and confirmed by comparative SDS/PAGE with known amounts of rhodopsin. Exchange of water in affinity-purified rhodopsin was accomplished by first drying with a Speed-Vac and then returning the sample to its original volume with 97% H₂O¹⁸ water (Cambridge Isotopes) with equilibration for 48 h prior.

Meta II Decay.

Meta II decay rate determinations were performed by the Trp fluorescence method (λ_excitation = 295 nm and λ_emission = 330 nm) of Farrens and Khorana (44). All measurements were performed with 30 nM Rho purified by 1D4 affinity chromatography (either exposed or not to radiolysis) dissolved in buffer consisting of 10 mM BTP, 100 mM NaCl, and 1 mM DDM, pH 6.0, that favors formation of Meta II. A Perkin-Elmer LS 55 Luminescence Spectrophotometer was used to measure the intrinsic fluorescence increase because of Trp residues, which correlates with the decrease in the protonated Schiff base concentration (44–47). Rhodopsin was bleached by a Fiber-Lite illuminator covered with a band-pass wavelength filter (480–520 nm) for 15 s immediately before the fluorescence measurements. Bleaching was carried out from a distance of 10 cm to prevent heat accumulation, and a thermostat was applied to stabilize the temperature of the cuvette at 20 °C. Fluorimeter slit settings were 5.0 nm at 295 nm for excitation and 10 nm at 330 nm for emission.

Radiolytic Labeling.

Protein samples in detergent were exposed to synchrotron X-ray white light at the National Synchrotron Light Source's (Brookhaven National Laboratory, Upton, NY) beamline X-28C operating at a ring energy of 2.8 GeV (15). The X-ray beam parameters were optimized by using the standard fluorophore assay; this assays monitors the loss of intensity of an Alexa fluorophore to determine the effective hydroxyl radical concentration (48, 49). At low flux density, the scavenging effects of detergent requires long exposure times (>100 ms) that permit secondary radical reactions and result in low signal-to-noise LC-MS data. The high X-ray flux density generated by focusing the beam with a mirror (mirror angle to 5.5 mrad and the bender value to 8.0 mm) (17, 49) permits a sufficient dose to be delivered in a few milliseconds, reducing chemical noise and enhancing LC-MS data acquisition. Rhodopsin samples were irradiated using time intervals raging from 1–10 ms with a continuous flow method using modified KinTek (KinTek Corporation) apparatus. Solvent exchange experiments were carried out by 1:1 (H₂O¹⁶ buffer:H₂O¹⁸ buffer) solvent mixing with 2–3 ms instrumental dead time followed by delays of 50, 100, 500, 5,000, and 30,000 ms before synchrotron beam exposure for 6- or 40-ms time intervals. These experiments were carried out in the KinTek quench flow mixer (KinTek Corporation) (48). All exposures were carried out at 4 °C, and to all samples methionine-amide (final concentration 10 mM) was added to quench any peroxide-induced or free radical-induced secondary oxidations during the postexposure period (50). Samples were frozen in dry ice and stored at −80 °C before proteolytic cleavage and liquid chromatography mass spectrometric (LC-MS) analyses. Details of these latter procedures are contained in SI Text.