Letter

pubs.acs.org/JPCL

Powdered G‑Protein-Coupled Receptors † § † § † ‡ Suchithranga M. D. C. Perera, , Udeep Chawla, , and Michael F. Brown*, , † Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States ‡ Department of Physics, University of Arizona, Tucson, Arizona 85721, United States

*S Supporting Information

ABSTRACT: Preparation and storage of functional membrane proteins such as G-protein-coupled receptors (GPCRs) are crucial to the processes of drug delivery and discovery. Here, we describe a method of preparing powdered GPCRs using rhodopsin as the prototype. We purified rhodopsin in CHAPS detergent with low detergent to protein ratio so the bulk of the sample represented protein (ca. 72% w/w). Our new method for generating powders of membrane proteins followed by rehydration paves the way for conducting functional and biophysical experiments. As an illustrative application, powdered rhodopsin was prepared with and without the cofactor 11-cis-retinal to enable partial rehydration of the protein with D2O in a controlled manner. Quasi-elastic neutron scattering studies using both spatial motion and energy landscape models form the basis for crucial insights into structural fluctuations and thermodynamics of GPCR activation.

embrane proteins comprise more than one-half of the protein properties and stability can be affected due to local − M pharmaceutical drug targets.1 3 The G-protein-coupled concentration of the salts of the buffer.9 When water crystals receptors (GPCRs) are particularly important because they are are formed during the freezing process, the hydrated salt ions involved in regulation of key physiological processes involving are partitioned into the excluded volume at very high mediation of cellular responses to hormones and neuro- concentrations, which in turn can affect the protein properties. 4 transmitters, including vision, taste, and olfaction. Studies of By contrast, flash freezing the protein using liquid nitrogen the structure and function of GPCRs are crucial for during lyophilization avoids such local concentration of salt development of more effective pharmaceuticals and for ions before sublimation of water to obtain the powdered 5 understanding the general mechanisms of cellular signaling. protein sample. Because the frozen water is removed by ffi One of the great di culties in studying membrane proteins sublimation, freeze-drying avoids the potentially detrimental such as GPCRs is their location within biological membranes. thawing of the sample, which leads to local concentration of Investigation of GPCRs typically requires extraction from their salts as in the case of conventional freezing. native lipid environment and functional and structural In the present application, we investigated whether stabilization by embedding them into membrane mimetics 6 membrane proteins such as rhodopsin can be prepared as dry like detergent protein-micelles, protein-detergent-lipid bi- powders with either lipids or detergents. The dry protein 7 − 8 celles, or protein lipid nanodiscs. The process of removal powders regain their photochemical functionality upon from the native physiological environment can be detrimental rehydration with bulk excess water. (The high optical density to membrane protein integrity and stability, even if the process makes it challenging to investigate whether rhodopsin can be is performed at low temperatures. Here, we report a novel light activated in the powdered form, i.e., before rehydration.) method of preserving solubilized fully functional GPCRs For this purpose, we removed the bulk lipids10 to purify the through a detergent lyophilization method, which potentially integral membrane protein rhodopsin (Figure 1a) in a increases their shelf life well beyond what one could achieve by detergent environment (for details see Supporting Information, mere freezing of the detergent-solubilized protein samples. This SI). We chose 3-[(3-cholamidopropyl)dimethylammonio]-1- new method to stabilize GPCRs forms the basis for a number propanesulfonate (CHAPS) detergent with an aggregation of applications, and paves the way for applying quasi-elastic 11 neutron scattering (QENS) methods to powdered rhodopsin. number of ca. 10 to achieve the minimum protein to Lyophilization (also known as freeze-drying) is a technique detergent molar ratio (27:1) (ca. 72% w/w protein) in the samples. Bovine retinal disk membranes (RDM) were of dehydrating a sample under a vacuum at cryogenic fi temperatures; the method converts a solution of a protein solubilized in CHAPS detergent, and puri ed using a zinc- 10 fi sample to a powder by removing water through the sublimation extraction method. The rhodopsin puri ed in CHAPS had a process. Obtaining the powdered protein sample in this way may enhance the stability of the protein. Moreover, this method Received: October 9, 2016 of protein preservation has additional advantages over freezing Accepted: October 10, 2016 the samples.9 For instance, during conventional freezing, the Published: October 10, 2016

© 2016 American Chemical Society 4230 DOI: 10.1021/acs.jpclett.6b02328 J. Phys. Chem. Lett. 2016, 7, 4230−4235 The Journal of Physical Chemistry Letters Letter

G-protein (transducin). Activation of the model GPCR rhodopsin is controlled by the chromophore in the ligand- binding pocket of the receptor. Upon photon absorption, the 11-cis-retinal isomerizes to all-trans, yielding rearrangement of the protein conformation by two protonation switches.28,29 Photoisomerization of retinal occurs within less than 200 fs,30 causing rhodopsin to undergo a series of multiscale conforma- tional transitions,31,32 where dynamics of the protein play a crucial role in its biological signaling function. Solid-state NMR methods,21,33 X-ray diffraction,34 solution X-ray scattering,35 and site-directed spin labeling (SDSL)36 have all been extensively applied to study the functional reaction cycle of rhodopsin. Current understanding of pharmacologically important GPCRs such as the serotonin, β-adrenergic, or angiotensin 5,37 − receptors suggests that protein dynamics hold the key to Figure 1. Powdered rhodopsin CHAPS detergent complex retains understanding their functions. Conformational fluctuations of functionality upon rehydration. (a) Dark-state rhodopsin crystal structure (1U19)39 overlaid on ligand-free opsin crystal structure the protein upon extracellular stimulation lead to the activation 52 − − of GPCRs in a cellular membrane lipid environment. Notably, (PDB code 3CAP). (b) UV visible spectra for rhodopsin CHAPS 38 complex after lyophilization and rehydration with excess H O X-ray crystallographic experiments and recent time-resolved 2 35 (keeping the detergent to protein molar ratio constant). Results are wide-angle X-ray scattering studies conducted on the shown for the dark state, upon light activation, and for fully bleached prototypical visual GPCR rhodopsin have revealed valuable opsin. (c) Far-UV circular dichroism spectra for powdered rhodopsin information about the conformational changes that occur and powdered opsin after rehydration in excess H2O. Upon during activation. X-ray crystal structures are presently available rehydration samples contained ca. 220 μM rhodopsin in ca. 6 mM 39 ff for rhodopsin in the dark state, as well as several freeze- CHAPS in 15 mM sodium phosphate bu er (pH 6.9), and spectra trapped photointermediates,38,40 including the ligand-free opsin were collected at 15 °C. apoprotein. However, thus far little information is available regarding how the internal dynamics of the protein change − 41 purity (A280/A500) ratio in the range of 1.7 1.8 and a yield of during GPCR activation. Application of the powdered GPCR about 60% (w/w) (percentage purified rhodopsin recovered preparation method together with use of neutron scattering from RDM (see SI for details). The nominal amount of techniques allows for the study of changes in the protein CHAPS detergent in the solid sample was about 28% (w/w), dynamics upon rhodopsin activation. Current findings suggest and was adjusted using ultracentrifugal filters of 30 kDa molar the intrinsic dynamics of the protein are unlocked by the light- mass cutoff. Thereafter, the protein−CHAPS detergent induced isomerization of the 11-cis-retinal cofactor needed for solution was subjected to lyophilization by flash freezing at interaction of the GPCR with its cognate G-protein (trans- 77 K. Surprisingly, UV−visible spectroscopic characterization of ducin).26 the powdered samples (after rehydrating) by dilution in excess As an illustration, both elastic and quasi-elastic neutron water, keeping the detergent to protein ratio constant (ca. 6 scattering methods have been applied to rhodopsin as a mM CHAPS and 220 μM protein), showed the photochemical canonical GPCR prototype.26 A combination of the novel functionality of rhodopsin remained unaffected by lyophiliza- powdered rhodopsin sample preparation and its application to tion (Figure 1b). The 500 nm peak observed for the rehydrated quasi-elastic neutron scattering (QENS)42 provides insights powdered rhodopsin−CHAPS samples indicated that the into the hydrogen-atom dynamics in dark-state rhodopsin rhodopsin was still in the dark state, and that photoillumination versus the ligand-free opsin apoprotein.26 Backscattering yielded a mixture of inactive metarhodopsin-I and active spectrometers such as BASIS42 open new possibilities and metarhodopsin-II states.12 This is surprisingly true for both opportunities of extending this technology to complex and rhodopsin in powdered disk membranes (Figure S1) and biologically relevant systems such as membrane proteins powdered rhodopsin−CHAPS detergent preparations upon (Figure 2). The new sample preparation method plus advances rehydration (Figure 1b). Moreover, far-UV circular dichroism in QENS technology were applied to directly probe the effect (CD) spectra acquired for the powdered rhodopsin−CHAPS of the retinal cofactor on the dynamics of rhodopsin in the β- samples and powdered opsin−CHAPS samples when redis- fluctuation time range crucial for its activation. For the QENS solved in water (ca. 220 μM protein) revealed that the helicity applications, protein samples are required with the exchange- − was indeed conserved as expected (Figure 1c).13 16 able protons replaced with deuterons and controlled hydration 17,18 Further applications potentially include drug design, dry of the samples with D2O. Therefore, it is imperative to remove powder inhaling,19 studies of GPCR signaling mechanisms,20 all the bulk water to produce dry rhodopsin samples via − solid state NMR21 23 and spin-label EPR24,25 spectroscopy, and lyophilization. As a control, we first lyophilized rhodopsin in neutron scattering studies.26 Here, we show the use of the native disk membranes (RDM) (see SI), and the resulting powdered GPCR preparation method to investigate rhodopsin lyophilized powder was resuspended with distilled water and versus the ligand-free opsin apoprotein. Rhodopsin is a class A characterized. We found that lyophilization does not affect the GPCR responsible for vision under dim light conditions in purity (A280/A500 spectral ratio) of the retinal disk membranes vertebrates. It is the canonical prototype of the Rhodopsin (RDM) (Figure S1). family of GPCRs. The chromophore 11-cis-retinal locks the Notably, for proteins the QENS method is used to study the rhodopsin in the inactive dark state,27 where it acts as an translational, rotational, and diffusive motions involving inverse agonist by preventing the interaction with its cognate hydrogen atoms, due to the polypeptide backbone, methylene,

4231 DOI: 10.1021/acs.jpclett.6b02328 J. Phys. Chem. Lett. 2016, 7, 4230−4235 The Journal of Physical Chemistry Letters Letter

how the dynamics of the protein were affected upon rhodopsin photoactivation. Protein dynamics of the dark-state rhodopsin were compared to those of ligand-free opsin,26 which is structurally similar to active metarhodopin-II.38 Previous neutron scattering studies conducted to unravel the local dynamics of bacteriorhodopsin and visual rhodopsin in the dark and light-activated states have revealed few significant differ- ences.46,47 Our optimization of the sample preparation (see above) for the QENS experiment and methods refinements48 made it possible to capture the elusive protein dynamics during rhodopsin activation, which was not possible previously. Neutron scattering spectra are conventionally interpreted by a spatial motion model (SMM) in terms of separate elastic and quasi-elastic processes.26 The QENS spectrum is separated into an elastic component (elastic line at ΔE = 0), and a quasi- elastic component (broadened wings of the spectrum) treated as a Lorentzian centered around ΔE = 0 (elastic line). In the SMM analysis, the broadening of the QENS spectrum is ascribed to spatial motion of the probed atoms. Application of β τ Figure 2. Increased temperature yields reduction in elastic scattering the SMM in the time domain yields -relaxation times ( β) that 26 with greater quasi-elastic broadening in QENS spectra. Normalized correspond to the local structural fluctuations of the protein. ω dynamic incoherent scattering function, Sinc(Q, ) plotted as a function Further analysis of the QENS spectra probes the relaxational of energy transfer (E) for dark-state rhodopsin at temperatures varying dynamics of hydrogen atoms in various length scales of the − from T = 220 to 300 K for Q = 1.1 Å 1. The QENS spectra were protein molecule, including vibrations, local β-fluctuations, and normalized to unity over the measured range of transfer energy from α − μ collective -processes. Applying the spatial motion model to 120 to +120 eV. The instrument resolution function was collected the QENS data obtained for the powdered dark-state rhodopsin at 10 K and is plotted in light blue. Inset: Expansion of elastic and ligand-free opsin samples suggests crucial insights into the component at various temperatures. Note that dark-state rhodopsin role of the retinal cofactor in regulating protein mobility.26 The has a reduction in the elastic scattering and increased quasi-elastic τ broadening of the spectral wings as temperature increases. SMM implies the characteristic β relaxation time calculated for the ligand-free opsin is substantially longer versus the dark-state rhodopsin. A possible explanation is that the opsin apoprotein 26 and methyl groups.43 The incoherent neutron scattering is softer than dark-state rhodopsin. Yet, Arrhenius plots reveal technique is optimal for studying the dynamics of biological the activation energies associated with the local fluctuations in 26 molecules, as they mainly contain hydrogen atoms, which have the dark-state rhodopsin and the ligand-free opsin are similar. the largest incoherent scattering cross section for neutrons.44 Differences between the relaxation rates can be explained by the ffi τ Quasi-elastic neutron scattering methods enable one to probe Arrhenius coe cient ( 0) (pre-exponential factor), which molecular motions from picoseconds up to nanoseconds within includes steric effects on the local hydrogen-atom dynamics. the atomic to molecular length scales.26 Upon hydrating the The slower relaxation rate in opsin also suggests the possibility protein sample with D2O, the results of a QENS experiment are of greater steric crowding, that retards the hydrogen-atom expressed as the dynamic structure factor, which is dominated dynamics within the β-relaxation time regime versus dark-state by the contributions from nonexchangeable hydrogen atoms in rhodopsin. Slowing down the hydrogen atom dynamics would the protein. The QENS data can be analyzed both in the energy be consistent with under-hydration of the opsin conformation domain by a model-free analysis and in the time domain by versus dark-state rhodopsin.26 This increased steric hindrance introduction of motional models. For instance, a spatial motion for local fluctuations in opsin is reflected by an increase in the τ model (SMM) can be introduced by applying mode-coupling pre-exponential factor ( 0), in the Arrhenius equation, yielding theory, as originally used to describe the complex dynamics in slower local dynamics in opsin versus dark-state rhodopsin. glass-forming liquids. A highly nonexponential relaxation of the Greater steric crowding by collapsing of the protein structure density correlations and single-particle correlation functions is could result from removal of the retinylidene cofactor for opsin thus observed. Alternatively, an energy landscape model (ELM) versus dark-state rhodopsin. Formation of opsin by dissociation can be introduced as described by Frauenfelder et al.,45 in of the all-trans retinal from metarhodopsin-II entails two forms: which there is no separation into elastic and quasi-elastic lines. the more open low-pH form (Ops*) with basal activity and the Rather, the entire spectrum is inhomogeneously broadened due closed inactive opsin (Ops) state. The Ops* form is similar to to a random walk of the protein among the various tiers of the the active metarhodopsin-II structure, whereas Ops is believed energy landscape, in which the neutrons are treated as de to be structurally similar to dark-state rhodopsin.27,49 A more Broglie wave packets.45 collapsed protein structure for opsin compared to dark-state Together with QENS techniques the novel powdered GPCR rhodopsin would be consistent with elevated mechanical preparation method opens the door to uncovering subtle rigidity of ligand-free apoprotein as reported previously.50 changes in the protein dynamics due to the retinal cofactor of Recently, the conventional separation into distinct elastic and 26 − rhodopsin. For D2O-hydrated powders of rhodopsin quasi-elastic bands has beencalledintoquestionby CHAPS detergent, the majority of the recorded signal is due Frauenfelder et al.,45 who instead propose an energy landscape to the protein dynamics, and the correction for the presence of model (ELM). The QENS spectrum is thus composed of the detergent is minimal during the subsequent data reduction multiple individual spectral lines corresponding to the and analysis. The QENS experiments were focused on studying conformational substates of the protein. The entire QENS

4232 DOI: 10.1021/acs.jpclett.6b02328 J. Phys. Chem. Lett. 2016, 7, 4230−4235 The Journal of Physical Chemistry Letters Letter spectrum is considered to be inhomogeneously broadened, for determine the level of purity (A280/A500 absorption ratio was example, by a random walk of the protein molecule among the typically 2.4). The rhodopsin concentration was estimated from various tiers in the energy landscape (EL) due to the the absorbance of the sample at 500 nm.29 The sample was conformational substates. This wave-mechanics-based model photobleached with green LED light of 515 nm, which activates 41,51 considers the incident neutrons as de Broglie wave packets that rhodopsin to form metarhodopsin-II. Including NH2OH exchange energy with the protein during their encounter. ensures complete photobleaching of the dark-state rhodopsin Because the energy exchange depends on the conformational and is confirmed by the nearly zero absorbance at ca. 500 nm substates, the ELM approach scans the free energy landscape of (Figure S1; see SI for further details on preparation and rhodopsin activation, as well as the structural fluctuations in the characterization of powdered RDM samples). protein. An increase of temperature broadens the quasi-elastic Preparation of CHAPS-Solubilized Protein Samples. Retinal spectrum (Figure 2), corresponding to an increase in the disk membranes (RDM) with a final rhodopsin concentration number of conformational substates occupied by the protein. of 400 μM were dissolved in 15 mM sodium phosphate buffer, Interpretation of the QENS spectra using the ELM45 also pH 6.9, containing 100 mM CHAPS and 100 mM zinc acetate supports a more collapsed protein structure in opsin (Ops) and incubated for 30 min at 4 °C.10 The hydrophobic part of versus the dark-state rhodopsin. The overall narrowing of the the detergent interacts with the protein hydrophobic surface, QENS spectra26 suggests a reduced ensemble of conforma- whereas the hydrophilic part interacts with the polar solvent tional substates that exchange energy with the incoming thus solubilizing the membrane protein. The solubilized neutron wave packets in ligand-free opsin versus dark-state rhodopsin was centrifuged at 24 000g (Sorvall SS-34 rotor) rhodopsin. for 30 min. Next, the solution was diluted with two volumes of In summary, a novel powdered GPCR sample preparation 15 mM sodium phosphate buffer, pH 6.9, and centrifuged at procedure in detergent has been developed for the first time. A 24 000g (Sorvall SS-34 rotor) for 30 min to remove high rhodopsin:detergent ratio was the key to the applications nonsolubilized membranes. The supernatant was characterized reported herein. The powdered GPCR−detergent complex using UV−visible spectroscopy to determine the quantity and preparation removes water from the samples completely and purity. The CHAPS-solubilized rhodopsin was diluted with 15 enables the subsequent controlled hydration with D2Oby mM sodium phosphate buffer, pH 6.9, and concentrated with isopiestic transfer through the vapor phase. The UV−visible 30 kDa-cutofffilters to adjust the detergent to rhodopsin molar characterization after rehydrating the powdered samples shows ratio to be 27:1 (72% w/w protein). To prepare the powdered that even one year after sample preparation, the proteins are opsin sample, 1% (w/v) hydroxylamine was added to 30 mg/ still photochemically functional. This new method of preparing mL rhodopsin in 50 mM CHAPS containing 15 mM sodium functional D2O-rehydrated powders of rhodopsin may be phosphate buffer solution, pH 6.9, and completely photo- applied to other GPCRs in the future, thus demonstrating the bleached using a 515 nm LED lamp before starting the proof of concept. An important question remaining for future lyophilization step. The presence of hydroxylamine ensures that research is whether the powdered GPCR method is applicable hydrolysis of the Schiff base linkage between retinal and Lys296 to other members of the Rhodopsin superfamily of G-protein- occurs upon photoillumination.51 The resulting hydrolysis of coupled receptors. the retinal from the rhodopsin yields the apoprotein, opsin.12 The detergent to opsin molar ratio was adjusted as described ■ MATERIALS AND METHODS above. About 300 mg (rhodopsin alone) was used for Powdered membrane protein samples containing 72% (w/w) preparation of the opsin sample. A total of ca. 1000 mg of of bovine rhodopsin and 28% (w/w) of CHAPS (3-[(3- rhodopsin in RDM was solubilized to obtain the powdered cholamidopropyl)dimethylammonio]-1-propanesulfonate) de- dark-state and opsin samples used for the neutron scattering tergent were prepared. About 600 mg of the powdered application.26 rhodopsin sample was divided into two separate containers, The protein samples were lyophilized as follows (for details one for preparing a dark-state rhodopsin sample and the other see SI). First, the CHAPS detergent-solubilized protein samples for the ligand-free opsin apoprotein. The opsin was prepared by were flash frozen at 77 K using liquid nitrogen. Next, the flash- photobleaching the dark-state sample with a locally constructed frozen samples were subjected to a vacuum of 100 mTorr for 515 nm LED light source. Far-UV circular dichroism (CD) 12 h in a homemade Schlenk line setup. This step was carried spectra were collected for the dark-state rhodopsin and opsin out in a dark room to avoid light exposure of the dark-state samples and confirmed that the helicity is conserved after the rhodopsin samples. Finally, after ca. 12 h the powdered protein lyophilization and rehydration (Figure 1c). The samples were samples were transferred into Falcon 15 mL centrifuge tubes − ° hydrated with D2O inside a glovebox, with a hydration level of for storage at 80 C. A portion of the powdered sample was ≈ − h 0.27 (g of D2O/g of protein) (ca. 600 water molecules per rehydrated with excess H2O for UV visible characterization. rhodopsin molecule) and were enclosed in aluminum foil to For the applications to quasi-elastic neutron scattering, it is prevent exposure to light. Finally, the samples were inserted in necessary to replace all the exchangeable protons with a rectangular aluminum sample holder for the near-back- deuterons in the protein sample. Powdered samples prepared scattering spectrometer (BASIS) used for the neutron as described above were rehydrated with D2O and lyophilized 26 scattering experiments. again. This step (lyophilization with D2O) was repeated 3 times Preparation of Retinal Disk Membranes. All procedures were to ensure the exchange of protons with deuterons. The final dry carried out under dim red light (11-W Bright Lab Universal powdered rhodopsin and opsin samples were hydrated to 27% ° Red Safelight bulb, CPM Delta1, Inc., Dallas, TX), at 4 C. (w/w) using 99.9% D2O (Sigma-Aldrich, St Louis, MO) in a fi Retinal disk membranes (RDM) containing rhodopsin were glovebag lled with argon gas by placing a warm 99.9% D2O isolated from bovine retinas as described.41 The RDM pellet bath inside. Finally, the hydrated sample was transferred to the was resuspended in 15 mM sodium phosphate buffer, pH 6.9, aluminum cell, screwed tight, and inserted into the near and characterized using UV−visible spectroscopy51 to backscattering spectrometer.

4233 DOI: 10.1021/acs.jpclett.6b02328 J. Phys. Chem. Lett. 2016, 7, 4230−4235 The Journal of Physical Chemistry Letters Letter ■ ASSOCIATED CONTENT (16) Litman, B. J. Ultraviolet circular dichroism of rhodopsin in disk membranes and detergent solution. Methods Enzymol. 1982, 81, 629− *S Supporting Information 633. The Supporting Information is available free of charge on the (17) Frokjaer, S.; Otzen, D. E. Protein drug stability: a formulation ACS Publications website at DOI: 10.1021/acs.jpclett.6b02328. challenge. Nat. Rev. Drug Discovery 2005, 4, 298−306. (PDF) (18) Franks, F. Freeze-drying of bioproducts: putting principles into practice. Eur. J. Pharm. Biopharm. 1998, 45, 221−229. ■ AUTHOR INFORMATION (19) Salnikova, M.; Varshney, D.; Shalaev, E. Heterogeneity of protein environments in frozen solutions and in the dried state. In Corresponding Author Lyophilized Biologics and Vaccines: Modality-Based Approaches; *E-mail: [email protected]. Varshney, D., Singh, M., Eds.; Springer: New York, 2015; pp 11−24. Author Contributions (20) Herrmann, R.; Heck, M.; Henklein, P.; Hofmann, K. P.; Ernst, § α S.M.D.C.P. and U.C. contributed equally to this work. O. P. Signal transfer from GPCRs to G proteins: role of the G N- terminal region in rhodopsin-transducin coupling. J. Biol. Chem. 2006, Notes − fi 281, 30234 3041. The authors declare no competing nancial interest. (21) Struts, A. V.; Salgado, G. F.; Brown, M. F. Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in ■ ACKNOWLEDGMENTS membrane activation of rhodopsin. Proc. Natl. Acad. Sci. U. S. A. This work was supported by the U.S. National Institutes of 2011, 108, 8263−8268. (22) Brown, M. F.; Struts, A. V. Structural dynamics of retinal in Health (EY12049 and EY026041). We thank P. Fromme, H. 2 Frauenfelder, O. Monti, and A. Struts for discussions, T. rhodopsin activation viewed by solid-state H NMR spectroscopy. In Knowles and M. Pitman for assistance with sample preparation, Advances in Biological Solid-State NMR: Proteins and Membrane-Active and K. Bao for constructing the actinic light source used for the Peptides; Separovic, F., Naito, A., Eds.; The Royal Society of Chemistry: London, 2014; pp 320−352. neutron scattering experiments. (23) Laage, S.; Tao, Y.; McDermott, A. E. Cardiolipin interaction with subunit c of ATP synthase: solid-state NMR characterization. ■ REFERENCES − Biochim. Biophys. Acta 2015, 1848, 260 265. (1) Drews, J. Drug discovery: a historical perspective. Science 2000, (24) Hussain, S.; Kinnebrew, M.; Schonenbach, N. S.; Aye, E.; Han, 287, 1960−1964. S. Functional consequences of the oligomeric assembly of proteo- (2) Hopkins, A. L.; Groom, C. R. The druggable genome. Nat. Rev. rhodopsin. J. Mol. Biol. 2015, 427, 1278−1290. Drug Discovery 2002, 1, 727−730. (25) Borbat, P. P.; Freed, J. H. Pulse dipolar electron spin resonance: (3) Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. How many drug distance measurements. In Structural Information from Spin-Labels and targets are there? Nat. Rev. Drug Discovery 2006, 5, 993−996. Intrinsic Paramagnetic Centres in the Biosciences; Timmel, R. C., Harmer, (4) Lagerström, M. C.; Schiöth, H. B. Structural diversity of G R. J., Eds.; Springer: Berlin/Heidelberg, 2013; pp 1−82. protein-coupled receptors and significance for drug discovery. Nat. (26) Shrestha, U. R.; Perera, S. M. D. C.; Bhowmik, D.; Chawla, U.; − Rev. Drug Discovery 2008, 7, 339 357. Mamontov, E.; Brown, M. F.; Chu, X.-Q. Quasi-elastic neutron (5) Rosenbaum, D. M.; Rasmussen, S. G. F.; Kobilka, B. K. The scattering reveals ligand-induced protein dynamics of a G-protein- structure and function of G-protein-coupled receptors. Nature 2009, coupled receptor. J. Phys. Chem. Lett. 2016, 7, 4130−4136. − 459, 356 363. (27) Piechnick, R.; Ritter, E.; Hildebrand, P. W.; Ernst, O. P.; (6) Sanders, C. R.; Sönnichsen, F. Solution NMR of membrane − Scheerer, P.; Hofmann, K. P.; Heck, M. Effect of channel mutations on proteins: practice and challenges. Magn. Reson. Chem. 2006, 44, S24 the uptake and release of the retinal ligand in opsin. Proc. Natl. Acad. S40. Sci. U. S. A. 2012, 109, 5247−5252. (7) Duc, N. M.; Du, Y.; Thorsen, T. S.; Lee, S. Y.; Zhang, C.; Kato, (28) Mahalingam, M.; Martínez-Mayorga, K.; Brown, M. F.; Vogel, R. H.; Kobilka, B. K.; Chung, K. Y. Effective application of bicelles for Two protonation switches control rhodopsin activation in membranes. conformational analysis of G protein-coupled receptors by hydrogen/ Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 17795−17800. deuterium exchange mass spectrometry. J. Am. Soc. Mass Spectrom. (29) Chawla, U.; Jiang, Y.; Zheng, W.; Kuang, L.; Perera, S. M. D. C.; 2015, 26, 808−817. Pitman, M. C.; Brown, M. F.; Liang, H. A usual G-protein-coupled (8) Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Self-assembly of receptor in unusual membranes. Angew. Chem. 2016, 128, 598−602. discoidal phospholipid bilayer nanoparticles with membrane scaffold (30) Schoenlein, R. W.; Peteanu, L. A.; Mathies, R. A.; Shank, C. V. proteins. Nano Lett. 2002, 2, 853−856. The first step in vision: femtosecond isomerization of rhodopsin. (9) Matejtschuk, P. Lyophilization of proteins. Methods Mol. Biol. − 2007, 368,59−72. Science 1991, 254, 412 415. (10) Okada, T.; Takeda, K.; Kouyama, T. Highly selective separation (31) Li, J.; Edwards, P. C.; Burghammer, M.; Villa, C.; Schertler, G. F. X. Structure of bovine rhodopsin in a trigonal crystal form. J. Mol. Biol. of rhodopsin from bovine rod outer segment membranes using − combination of divalent cation and alkyl(thio)glucoside. Photochem. 2004, 343, 1409 1438. Photobiol. 1998, 67, 495−499. (32) Okada, T.; Ernst, O. P.; Palczewski, K.; Hofmann, K. P. (11) Jastrzebska, B.; Maeda, T.; Zhu, L.; Fotiadis, D.; Filipek, S.; Activation of rhodopsin: new insights from structural and biochemical − Engel, A.; Stenkamp, R. E.; Palczewski, K. Functional characterization studies. Trends Biochem. Sci. 2001, 26, 318 324. of rhodopsin monomers and dimers in detergents. J. Biol. Chem. 2004, (33) Kimata, N.; Pope, A.; Eilers, M.; Opefi, C. A.; Ziliox, M.; 279, 54663−54675. Hirshfeld, A.; Zaitseva, E.; Vogel, R.; Sheves, M.; Reeves, P. J.; Smith, (12) Ernst, O. P.; Bartl, F. J. Active states of rhodopsin. S. O. Retinal orientation and interactions in rhodopsin reveal a two- ChemBioChem 2002, 3, 968−974. stage trigger mechanism for activation. Nat. Commun. 2016, 7, 12683. (13) Shichi, H. Biochemistry of Vision; Academic Press: Waltham, MA, (34) Okada, T.; Le Trong, I.; Fox, B. A.; Behnke, C. A.; Stenkamp, R. 2012. E.; Palczewski, K. X-Ray diffraction analysis of three-dimensional (14) Shichi, H. Biochemistry of Visual Pigments. J. Biol. Chem. 1971, crystals of bovine rhodopsin obtained from mixed micelles. J. Struct. 246, 6178−6182. Biol. 2000, 130,73−80. (15) Albert, A. D.; Litman, B. J. Independent structural domains in (35) Malmerberg, E.; Bovee-Geurts, P. H. M.; Katona, G.; Deupi, X.; the membrane protein bovine rhodopsin. Biochemistry 1978, 17, Arnlund, D.; Wickstrand, C.; Johansson, L. C.; Westenhoff, S.; 3893−3900. Nazarenko, E.; Schertler, G. F. X.; Menzel, A.; de Grip, W. J.; Neutze,

4234 DOI: 10.1021/acs.jpclett.6b02328 J. Phys. Chem. Lett. 2016, 7, 4230−4235 The Journal of Physical Chemistry Letters Letter

R. Conformational activation of visual rhodopsin in native disc membranes. Sci. Signaling 2015, 8, ra26. (36) McCoy, J.; Hubbell, W. L. High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1331−1336. (37) Kruse, A. C.; Kobilka, B. K.; Gautam, D.; Sexton, P. M.; Christopoulos, A.; Wess, J. Muscarinic acetylcholine receptors: novel opportunities for drug development. Nat. Rev. Drug Discovery 2014, 13, 549−560. (38) Choe, H. W.; Kim, Y. J.; Park, J. H.; Morizumi, T.; Pai, E. F.; Krauß, N.; Hofmann, K. P.; Scheerer, P.; Ernst, O. P. Crystal structure of metarhodopsin II. Nature 2011, 471, 651−655. (39) Okada, T.; Sugihara, M.; Bondar, A.-N.; Elstner, M.; Entel, P.; Buss, V. The retinal conformation and its environment in rhodopsin in light of a new 2.2 Å crystal structure. J. Mol. Biol. 2004, 342, 571−83. (40) Standfuss, J.; Edwards, P. C.; D’Antona, Å.; Fransen, M.; Xie, G.; Oprian, D. D.; Schertler, G. F. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 2011, 471, 656− 660. (41) Struts, A. V.; Chawla, U.; Perera, S. M. D. C.; Brown, M. F. Investigation of rhodopsin dynamics in its signaling state by solid-state deuterium NMR spectroscopy. Methods Mol. Biol. 2015, 1271, 133− 158. (42) Mamontov, E.; Herwig, K. W. A time-of-flight backscattering spectrometer at the Spallation Neutron Source, BASIS. Rev. Sci. Instrum. 2011, 82, 085109. (43) Lagi, M.; Baglioni, P.; Chen, S.-H. Logarithmic decay in single- particle relaxation of hydrated lysozyme powder. Phys. Rev. Lett. 2009, 103, 108102. (44) Chu, X.-q.; Mamontov, E.; O’Neill, H.; Zhang, Q. Apparent decoupling of the dynamics of a protein from the dynamics of its aqueous solvent. J. Phys. Chem. Lett. 2012, 3, 380−385. (45) Frauenfelder, H.; Fenimore, P. W.; Young, R. D. A wave- mechanical model of incoherent quasi-elastic scattering in complex systems. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 12764−12768. (46) Fitter, J.; Verclas, S. A. W.; Lechner, R. E.; Büldt, G.; Ernst, O. P.; Hofmann, K. P.; Dencher, N. A. Bacteriorhodopsin and rhodopsin studied by incoherent neutron scattering: dynamical properties of ground states and light activated intermediates. Phys. B 1999, 266,35− 40. (47) Svergun, D. I.; Richard, S.; Koch, M. H. J.; Sayers, Z.; Kuprin, S.; Zaccai, G. Protein hydration in solution: experimental observation by x-ray and neutron scattering. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 2267−2272. (48) Chu, X.-q.; Mamontov, E.; O’Neill, H.; Zhang, Q. Temperature dependence of logarithmic-like relaxational dynamics of hydrated tRNA. J. Phys. Chem. Lett. 2013, 4, 936−942. (49) Vogel, R.; Siebert, F. Conformations of the active and inactive states of opsin. J. Biol. Chem. 2001, 276, 38487−38493. (50) Kawamura, S.; Gerstung, M.; Colozo, A. T.; Helenius, J.; Maeda, A.; Beerenwinkel, N.; Park, P. S.; Müller, D. J. Kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin. Structure 2013, 21, 426−437. (51) Brown, M. F. UV−visible and infrared methods for investigating lipid−rhodopsin membrane interactions. Methods Mol. Biol. 2012, 914, 127−153. (52) Park, J. H.; Scheerer, P.; Hofmann, K. P.; Choe, H. W.; Ernst, O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 2008, 454, 183−187.

4235 DOI: 10.1021/acs.jpclett.6b02328 J. Phys. Chem. Lett. 2016, 7, 4230−4235