Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

A Proteorhodopsin-Related Photosensor Expands the Repertoire of Structural Motifs Employed by Sensory Rhodopsins.

Microbial rhodopsins are light-activated retinal-binding membrane proteins that perform a variety of ion transport and photosensory functions. They display several cases of convergent evolution where the same function is present in unrelated or very distant protein groups. Here we report another possible case of such convergent evolution, describing the biophysical properties of a new group of sensory rhodopsins. The first representative of this group was identified in 2004 but none of the members had been expressed and characterized. The well-studied haloarchaeal sensory rhodopsins interacting with methyl-accepting Htr transducers are close relatives of the halobacterial proton pump bacteriorhodopsin. In contrast, the sensory rhodopsins we describe here are relatives of proteobacterial proton pumps, proteorhodopsins, but appear to interact with Htr-like transducers likewise, even though they do not conserve the residues important for the interaction of haloarchaeal sensory rhodopsins with their transducers. The new sensory rhodopsins display many unusual amino acid residues, including those around the retinal chromophore; most strikingly, a tyrosine in place of a carboxyl counterion of the retinal Schiff base on helix C. To characterize their unique sequence motifs, we augment the spectroscopy and biochemistry data by structural modeling of the wild-type and three mutants. Taken together, the experimental data, bioinformatics sequence analyses, and structural modeling suggest that the tyrosine/aspartate complex counterion contributes to a complex water-mediated hydrogen-bonding network that couples the protonated retinal Schiff base to an extracellular carboxylic dyad.

Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K+ and Na+ selectivities.

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K+ over Na+ in the absence of the canonical tetrameric K+ selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na+ channel with >100-fold larger Na+ to K+ permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K+ versus Na+ selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating.

Structural Foundations of Potassium Selectivity in Channelrhodopsins.

Potassium-selective channelrhodopsins (KCRs) are light-gated K+ channels recently found in the stramenopile protist Hyphochytrium catenoides. When expressed in neurons, KCRs enable high-precision optical inhibition of spiking (optogenetic silencing). KCRs are capable of discriminating K+ from Na+ without the conventional K+ selectivity filter found in classical K+ channels. The genome of H. catenoides also encodes a third paralog that is more permeable for Na+ than for K+. To identify structural motifs responsible for the unusual K+ selectivity of KCRs, we systematically analyzed a series of chimeras and mutants of this protein. We found that mutations of three critical residues in the paralog convert its Na+-selective channel into a K+-selective one. Our characterization of homologous proteins from other protists (Colponema vietnamica, Cafeteria burkhardae, and Chromera velia) and metagenomic samples confirmed the importance of these residues for K+ selectivity. We also show that Trp102 and Asp116, conserved in all three H. catenoides paralogs, are necessary, although not sufficient, for K+ selectivity. Our results provide the foundation for further engineering of KCRs for optogenetic needs. IMPORTANCE Recently discovered microbial light-gated ion channels (channelrhodopsins) with a higher permeability for K+ than for Na+ (potassium-selective channelrhodopsins [kalium channelrhodopsins, or KCRs]) demonstrate an alternative K+ selectivity mechanism, unrelated to well-characterized "selectivity filters" of voltage- and ligand-gated K+ channels. KCRs can be used for optogenetic inhibition of neuronal firing and potentially for the development of gene therapies to treat neurological and cardiovascular disorders. In this study, we identified structural motifs that determine the K+ selectivity of KCRs that provide the foundation for their further improvement as optogenetic tools.

Unusual Photoisomerization Pathway in a Near-Infrared Light Absorbing Enzymerhodopsin.

Microbial and animal rhodopsins possess retinal chromophores which capture light and normally photoisomerize from all-trans to 13-cis and from 11-cis to all-trans-retinal, respectively. Here, we show that a near-infrared light-absorbing enzymerhodopsin from Obelidium mucronatum (OmNeoR) contains the all-trans form in the dark but isomerizes into the 7-cis form upon illumination. The photoproduct (λmax = 372 nm; P372) possesses a deprotonated Schiff base, and the system exhibits a bistable nature. The photochemistry of OmNeoR was arrested at <270 K, indicating the presence of a potential barrier in the excited state. Formation of P372 is accompanied by protonation changes of protonated carboxylic acids and peptide backbone changes of an α-helix. Photoisomerization from the all-trans to 7-cis retinal conformation rarely occurs in any solvent and protein environments; thus, the present study reports on a novel photochemistry mediated by a microbial rhodopsin, leading from the all-trans to 7-cis form selectively.

Biophysical characterization of light-gated ion channels using planar automated patch clamp.

Channelrhodopsins (ChRs) are proteins that guide phototaxis in protists and exhibit light-gated channel conductance when their genes are heterologously expressed in mammalian cells. ChRs are widely used as molecular tools to control neurons and cardiomyocytes with light (optogenetics). Cation- and anion-selective ChRs (CCRs and ACRs, respectively) enable stimulation and inhibition of neuronal activity by depolarization and hyperpolarization of the membrane, respectively. More than 400 natural ChR variants have been identified so far, and high-throughput polynucleotide sequencing projects add many more each year. However, electrophysiological characterization of new ChRs lags behind because it is mostly done by time-consuming manual patch clamp (MPC). Here we report using a high-throughput automated patch clamp (APC) platform, SyncroPatch 384i from Nanion Technologies, for ChR research. We find that this instrument can be used for determination of the light intensity dependence and current-voltage relationships in ChRs and discuss its advantages and limitations.

Bidirectional Photochemistry of Antarctic Microbial Rhodopsin: Emerging Trend of Ballistic Photoisomerization from the 13-cis Resting State.

The decades-long ultrafast examination of nearly a dozen microbial retinal proteins, ion pumps, and sensory photoreceptors has not identified structure-function indicators which predict photoisomerization dynamics, whether it will be sub-picosecond and ballistic or drawn out with complex curve-crossing kinetics. Herein, we report the emergence of such an indicator. Using pH control over retinal isomer ratios, photoinduced transient absorption is recorded in an inward proton pumping Antarctic microbial rhodopsin (AntR) for 13-cis and all-trans retinal resting states. The all-trans fluorescent state decays with 1 ps exponential kinetics. In contrast, in 13-cis it decays within ∼300 fs accompanied by continuous spectral evolution, indicating ballistic internal conversion. The coherent wave packet nature of 13-cis isomerization in AntR matches published results for bacteriorhodopsin (BR) and Anabaena sensory rhodopsin (ASR), which also accommodate both all-trans and 13-cis retinal resting states, marking the emergence of a first structure-photodynamics indicator which holds for all three tested pigments.

Kalium channelrhodopsins are natural light-gated potassium channels that mediate optogenetic inhibition.

Channelrhodopsins are used widely for optical control of neurons, in which they generate photoinduced proton, sodium or chloride influx. Potassium (K+) is central to neuron electrophysiology, yet no natural K+-selective light-gated channel has been identified. Here, we report kalium channelrhodopsins (KCRs) from Hyphochytrium catenoides. Previously known gated potassium channels are mainly ligand- or voltage-gated and share a conserved K+-selectivity filter. KCRs differ in that they are light-gated and have independently evolved an alternative K+ selectivity mechanism. The KCRs are potent, highly selective of K+ over Na+, and open in less than 1 ms following photoactivation. The permeability ratio PK/PNa of 23 makes H. catenoides KCR1 (HcKCR1) a powerful hyperpolarizing tool to suppress excitable cell firing upon illumination, demonstrated here in mouse cortical neurons. HcKCR1 enables optogenetic control of K+ gradients, which is promising for the study and potential treatment of potassium channelopathies such as epilepsy, Parkinson's disease and long-QT syndrome and other cardiac arrhythmias.

Conserved hydrogen-bond motifs of membrane transporters and receptors.

Membrane transporters and receptors often rely on conserved hydrogen bonds to assemble transient paths for ion transfer or long-distance conformational couplings. For transporters and receptors that use proton binding and proton transfer for function, inter-helical hydrogen bonds of titratable protein sidechains that could change protonation are of central interest to formulate hypotheses about reaction mechanisms. Knowledge of hydrogen bonds common at sites of potential interest for proton binding could thus inform and guide studies on functional mechanisms of protonation-coupled membrane proteins. Here we apply graph-theory approaches to identify hydrogen-bond motifs of carboxylate and histidine sidechains in a large data set of static membrane protein structures. We find that carboxylate-hydroxyl hydrogen bonds are present in numerous structures of the dataset, and can be part of more extended H-bond clusters that could be relevant to conformational coupling. Carboxylate-carboxyamide and imidazole-imidazole hydrogen bonds are represented in comparably fewer protein structures of the dataset. Atomistic simulations on two membrane transporters in lipid membranes suggest that many of the hydrogen bond motifs present in static protein structures tend to be robust, and can be part of larger hydrogen-bond clusters that recruit additional hydrogen bonds.

Light-driven proton transfers and proton transport by microbial rhodopsins - A biophysical perspective.

In the last twenty years, our understanding of the rules and mechanisms for the outward light-driven proton transport (and underlying proton transfers) by microbial rhodopsins has been changing dramatically. It transitioned from a very detailed atomic-level understanding of proton transport by bacteriorhodopsin, the prototypical proton pump, to a confounding variety of sequence motifs, mechanisms, directions, and modes of transport in its newly found homologs. In this review, we will summarize and discuss experimental data obtained on new microbial rhodopsin variants, highlighting their contribution to the refinement and generalization of the ideas crystallized in the previous century. In particular, we will focus on the proton transport (and transfers) vectoriality and their structural determinants, which, in many cases, remain unidentified.

Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity. IMPORTANCE Channelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study, we used manual and automated patch clamp recording of representative members of four channelrhodopsin families, including a family in dinoflagellates that we report in this study. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

Self-Organized Amphiphiles Are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments.

Analysis of membrane protein topography using fast photochemical oxidation of proteins (FPOP) has been reported in recent years but is still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. We found no significant change in bulk solvent radical scavenging activity upon the addition of disrupted cellular membranes up to 25600 cells/µL using an inline radical dosimeter. We confirmed the nonscavenging nature of the membrane in bulk solution with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration is a potent hydroxyl radical scavenger, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. Examination of both an extracellular peptide of the integral membrane protein bacteriorhodopsin as well as a novel hydroxyl radical dosimeter tethered to a Triton X-series amphiphile indicate that proximity to the membrane surface greatly decreases reaction with hydroxyl radicals, even though the oxidation target is equally solvent accessible. These results suggest that the observed reduced oxidation of solvent-accessible surfaces of integral membrane proteins is due to the high local concentration of radical scavengers in the membrane or membrane mimetics competing for the local concentration of hydroxyl radicals.

RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption.

Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulea and the other from haptophyte algae. Four closely related labyrinthulea ACRs, named RubyACRs here, exhibit a unique retinal-binding pocket that creates spectral sensitivities with maxima at 590 to 610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins thus far reported. We identified three spectral tuning residues critical for the red-shifted absorption. Photocurrents recorded from the RubyACR from Aurantiochytrium limacinum (designated AlACR1) under single-turnover excitation exhibited biphasic decay, the rate of which was only weakly voltage dependent, in contrast to that in previously characterized cryptophyte ACRs, indicating differences in channel gating mechanisms between the two ACR families. Moreover, in A. limacinum we identified three ACRs with absorption maxima at 485, 545, and 590 nm, indicating color-sensitive photosensing with blue, green, and red spectral variation of ACRs within individual species of the labyrinthulea family. We also report functional energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs.

The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants.

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

Mechanism of Inward Proton Transport in an Antarctic Microbial Rhodopsin.

Although the outward-directed proton transport across biological membranes is well studied and its importance for bioenergetics is clearly understood, inward-directed light-driven proton pumping by microbial rhodopsins has remained a mystery both physiologically and mechanistically. A new family of Antarctic rhodopsins, which is a subgroup within a novel class of schizorhodopsins reported recently, includes a member, denoted as AntR, which proved amenable to extensive characterization with experiments and computation. Phylogenetic analyses identify AntR as distinct from the well-studied microbial rhodopsins that function as outward-directed ion pumps, and bioinformatics sequence analyses reveal amino acid substitutions at conserved sites essential for outward proton pumping. Modeling and numerical simulations of AntR, combined with advanced analyses using the graph theory and centrality measures from social sciences, identify the dynamic three-dimensional network of hydrogen-bonded water molecules and amino acid residues that function as communication hubs in AntR. This network undergoes major rearrangement upon retinal isomerization, showing important changes in the connectivity of the active center, retinal Schiff base, to the opposing sides of the membrane, as required for proton transport. Numerical simulations and experimental studies of the photochemical cycle of AntR by spectroscopy and site-directed mutagenesis allowed us to identify pathways that could conduct protons in the direction opposite to that commonly known for outward-directed pumps.

Identifying lipids tightly bound to an integral membrane protein.

Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear. Here, we use solid-state nuclear magnetic resonance (SSNMR) spectroscopy and mass spectrometry to characterize these molecules. Numerous correlations atypical for protein amino acids were found and assigned in the SSNMR spectra. The chemical shift patterns correspond to N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose which are part of the Enterobacterial Common Antigen (ECA). These sugars undergo rapid anisotropic motions and are likely linked flexibly to a rigid anchor that tightly binds ASR. Phosphorus NMR reveals several signals that are characteristic of monophosphates, further suggesting phosphatidylglyceride as the ECA lipid carrier which is anchored to ASR. In addition, NMR signals corresponding to common phospholipid phosphatidylethanolamine (PE) have been detected. The presence of PE tightly interacting with ASR was confirmed using liquid chromatography-mass spectrometry. This article commemorates Professor Michèle Auger and her contributions to membrane biophysics and Nuclear Magnetic Resonance.

Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae.

Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. "Bacteriorhodopsin-like" cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR.

Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy.

Bridge: A Graph-Based Algorithm to Analyze Dynamic H-Bond Networks in Membrane Proteins.

Membrane proteins that function as transporters or receptors must communicate with both sides of the lipid bilayer in which they sit. This long distance communication enables transporters to move protons or other ions and small molecules across the bilayer and receptors to transmit an external signal to the cell. Hydrogen bonds, hydrogen-bond networks, and lipid-protein interactions are essential for the motions and functioning of the membrane protein and, consequently, of outmost interest to structural biology and numerical simulations. We present here Bridge, an algorithm tailored for efficient analyses of hydrogen-bond networks in membrane transporter and receptor proteins. For channelrhodopsin, a membrane protein whose functioning involves proton-transfer reactions, Bridge identifies extensive networks of protein-water hydrogen bonds and an unanticipated network that can bridge transiently two proton donors across a distance of ∼20 Å. Graphs of the protein hydrogen bonds reveal rapid propagation of structural changes within hydrogen-bond networks of mutant transporters and identify protein groups potentially important for the proton transfer activity. The algorithm is made available as a plugin for PyMol.

Solid-state NMR spectroscopy based atomistic view of a membrane protein unfolding pathway.

Membrane protein folding, structure, and function strongly depend on a cell membrane environment, yet detailed characterization of folding within a lipid bilayer is challenging. Studies of reversible unfolding yield valuable information on the energetics of folding and on the hierarchy of interactions contributing to protein stability. Here, we devise a methodology that combines hydrogen-deuterium (H/D) exchange and solid-state NMR (SSNMR) to follow membrane protein unfolding in lipid membranes at atomic resolution through detecting changes in the protein water-accessible surface, and concurrently monitoring the reversibility of unfolding. We obtain atomistic description of the reversible part of a thermally induced unfolding pathway of a seven-helical photoreceptor. The pathway is visualized through SSNMR-detected snapshots of H/D exchange patterns as a function of temperature, revealing the unfolding intermediate and its stabilizing factors. Our approach is transferable to other membrane proteins, and opens additional ways to characterize their unfolding and stabilizing interactions with atomic resolution.