Journal of Magnetic Resonance xxx (2015) xxx–xxx

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Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin Meaghan E. Ward, Leonid S. Brown, Vladimir Ladizhansky ⇑ Department of Physics and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario N1G 2W1, Canada

a r t i c l e

i n f o

Article history: Received 17 October 2014 Available online xxxx Keywords: Membrane proteins Protein structure Oligomerization Paramagnetic relaxation Dynamics

a b s t r a c t Studies of the structure, dynamics, and function of membrane proteins (MPs) have long been considered one of the main applications of solid-state NMR (SSNMR). Advances in instrumentation, and the plethora of new SSNMR methodologies developed over the past decade have resulted in a number of high-resolution structures and structural models of both bitopic and polytopic a-helical MPs. The necessity to retain lipids in the sample, the high proportion of one type of secondary structure, differential dynamics, and the possibility of local disorder in the loop regions all create challenges for structure determination. In this Perspective article we describe our recent efforts directed at determining the structure and functional dynamics of Anabaena Sensory Rhodopsin, a heptahelical transmembrane (7TM) protein. We review some of the established and emerging methods which can be utilized for SSNMR-based structure determination, with a particular focus on those used for ASR, a bacterial protein which shares its 7TM architecture with G-protein coupled receptors. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The knowledge of protein structure is key to understanding the detailed mechanisms of protein function. Although many new protein structures are deposited daily into the protein data bank, only a small fraction of those represent membrane proteins. Due to their amphipathic nature and sensitivity to the surrounding environment, which often make purification and subsequent crystallization a challenging task, structure determination of membrane proteins is a formidable challenge in structural biology. Solid-state NMR (SSNMR) is a rapidly developing technique which provides unique opportunities for the study of membrane proteins. Whereas solution NMR and X-ray crystallography are limited by the necessity of fast tumbling rate and ability to form large 3D crystals, respectively, SSNMR allows for the study of membrane proteins of any size in a lipid milieu which more closely mimics the cellular membrane environment. Many of the first SSNMR structures were obtained using oriented sample (OS) [1,2] approaches, and were those of small a-helical proteins. However, the accessibility of high magnetic fields and recent advances in probe technology and NMR methodologies have enabled the detailed structural and dynamics characterization of ⇑ Corresponding author. E-mail address: [email protected] (V. Ladizhansky).

multi-spanning membrane proteins using both OS and magic angle spinning (MAS) SSNMR. Microbial rhodopsins have traditionally acted as a testing ground in membrane protein research. In particular, the first structure of a membrane protein determined by electron microscopy in the year 1975 was that of an Archaeal proton pump, bacteriorhodopsin (BR) [3]. Because BR shares its seven transmembrane-helical (7TM) architecture with G-protein coupled receptors (GPCRs), its structure had been extensively used as a template to model GPCRs, until the first crystallographic study of visual rhodopsin was published in 2000 [4]. Microbial rhodopsins were also studied in some of the first applications of solution NMR to membrane proteins [5,6]. Likewise, many solid-state NMR methodologies have been developed on and applied to BR, from early applications of rotational resonance (R2) [7] and dipolar tensor correlation methods [8] to the determination of retinal conformation in selectively isotopically labeled samples, to the demonstration of high proton resolution in polytopic alpha-helical membrane proteins [9], to pioneering studies of the BR function using Dynamic Nuclear Polarization (DNP) [10]. Several other rhodopsins, including proteorhodopsin (PR) [11,12] sensory rhodopsin II (SRII) [13], Leptosphaeria rhodopsin [14], and Anabaena Sensory Rhodopsin [15] have also been structurally characterized using solid-state NMR. MAS SSNMR studies of large alpha-helical membrane proteins pose a significant challenge for several reasons. The lipid

http://dx.doi.org/10.1016/j.jmr.2014.11.017 1090-7807/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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environment complicates sample preparation and leads to an overall reduction in the amount of protein present in the sample, and thus a reduction in experimental sensitivity. The high molecular weight of proteins also reduces sensitivity, and leads to poor spectral resolution due to the high repetitiveness of hydrophobic residues, the dominance of a-helical secondary structure, and high spectral congestion, thus necessitating the use of less sensitive experiments of high dimensionality. Peripheral solvent-exposed regions of membrane proteins are often mobile and may be disordered, which creates additional sensitivity challenges for the detection of these regions. The applicability of proton-detection methods, which was recently demonstrated for bitopic helical proteins, and proteins of b-barrel structure [9,16], is limited in alphahelical bundles by incomplete backbone exchange [9,17]. Thus, the majority of applications of SSNMR to membrane proteins to date have relied on traditional methods employing carbon detection and robust polarization transfer schemes. In this Perspective we illustrate the challenges faced in the structure determination of a membrane protein, using the example of Anabaena Sensory Rhodopsin (ASR), one of the two 7TM proteins whose structure has recently been determined (the other structure is that of a G-protein coupled receptor, CXCR1, solved using rotational alignment by the Opella group [18]). ASR is a cyanobacterial light-sensitive receptor which is likely responsible for chromatic adaptation in its host cell [19,20]. ASR is unique among the known microbial rhodopsins in that it is believed to be the only rhodopsin to interact with a cytoplasmic soluble transducer (ASRT) (Fig. 1) [19,21,22]. ASR can be overexpressed in Escherichia coli, and reconstituted in lipids at a high protein-to-lipid ratio in a fully functional state. It forms stable trimers in both detergents and lipids and recent evidence suggests that ASR forms a two-dimensional lattice in DMPC:DMPA lipids (unpublished results). These properties are important for establishing high spectral resolution and sensitivity in ASR, which facilitates its structural and dynamics characterization. 2. Spectroscopic assignments Obtaining spectroscopic assignments is a prerequisite for sitespecific studies of protein structure, dynamics, and interactions.

Fig. 1. Cartoon representation of the signal transduction cascade of Anabaena Sensory Rhodopsin (ASR). ASR, shown as a seven-helical bundle embedded in the bilayer, with retinal (red) covalently linked to seventh helix G, interacts with a soluble tetrameric transducer (ASRT) and is believed to be responsible for the regulation of the genes of several light harvesting proteins. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

In and of itself, chemical shift data allow for initial secondary structure and torsion angle analysis to be performed using empirical methods such as chemical shift indexing or TALOS [23,24]. The successful application of assignment strategies discussed below depends on the available spectral resolution, determined by the structural homogeneity of the protein sample and by resonance dispersion. Currently, a large and growing number of membrane proteins of various classes have been shown to give highly resolved spectra [25]. Thus, preparation of structurally homogenous samples of membrane proteins does not appear to be a significant limitation for their structural characterization, provided that the protein can be expressed at sufficient levels. On the other hand, resonance dispersion is an intrinsic property of a molecule and is generally poorer in proteins with strong alpha-helical content. Indeed, large transmembrane regions generally have a high proportion of hydrophobic residues. These residues exist in structurally similar environments and are therefore likely to have similar chemical shifts. Nevertheless, interhelical interactions and helix packing create a sufficient number of distortions from ideal helicity which, in combination with variations in the electrostatic environment, improve the chemical shift dispersion in polytopic membrane proteins. In ASR particularly, there is a larger proportion of hydrophilic residues, which also contributes to better chemical shift dispersion. Due to the large number of peaks and the high occurrence of overlap the use of three-dimensional correlation spectroscopy is imperative, and needs to be assisted by simplifying labeling strategies. The minimalistic suite of assignment experiments requires three types of 3D correlation experiments, CANCO, NCACX, and NCOCX (Fig. 2), which allow for the unique identification of spin systems and linkage of these systems into contiguous fragments. In the NCACX and NCOCX experiments, backbone and side-chain atoms are correlated through DARR [26,27] mixing. In order to obtain both short and long intra-residue correlations, experiments with both short (20 ms for NCACX and 50 ms for NCOCX) and long (50 ms for NCACX and 100 ms for NCOCX) mixing times are collected. Short mixing time experiments generally yield one bond N[i]–Ca[i]–CO[i] and N[i]–Ca[i]–Cb[i], and in some cases two bond N[i]–Ca[i]–Cc[i], correlations in the NCACX and one bond N[i]– CO[i  1]–Ca[i  1], and in some cases two bond N[i]–CO[i  1]– Cb[i  1], correlations in the NCOCX. Longer mixing times result in three or four bond carbon–carbon connectivities between side chain atoms, as well as in inter-residue correlations of the N[i]– CO[i  1]–Ca[i] type. Further side-chain assignments can be obtained from high-sensitivity two dimensional carbon–carbon spectra. In Fig. 3 we show representative 2D planes of the 3D experiments recorded on ASR, demonstrating the typically high resolution that can be achieved in these experiments. The amino acid systems obtained from each experiment are highlighted in Fig. 2B. These systems are linked to each other through matching the chemical shifts of the side chain atoms, resulting in longer contiguous fragments. The correct and complete assignment of side chain correlations in the NCACX and NCOCX experiments is essential for the identification of amino acid type and reliable linking of spin systems. As backbone resonances are assigned and amino acid types determined the linked resonances are mapped to unique fragments in the protein sequence and specific resonance assignments are obtained. Using these methods alone we were able to assign backbone and side-chain resonances for 169 residues in a single sample of uniformly 15N,13C-labeled ASR [28]. However, there were a large number of incomplete spin systems detected that either lacked critical backbone resonances, could not be identified by the amino acid type, or could not be reliably linked with other spin systems. In particular, aromatic residues were difficult to identify due to the lower intensity of their side chain resonances.

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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Fig. 2. Solid-state NMR assignment strategies. (A) N[i]–CO[i  1]–CX[i  1], Ca[i]–N[i]–CO[i  1] and N[i]–Ca[i]–CX[i] correlations are obtained from the three-dimensional NCOCX, CANCO, and NCACX experiments, respectively. By matching overlapping pairs of resonances (for example N[i]–CO[i  1] in both NCOCX and CANCO, or N[i]–Ca[i] in the NCACX and CANCO) the extended spin systems shown in B can be built from these experiments. The extended spin systems can similarly be linked into contiguous fragments by matching the shifts of backbone and side chain atoms indicated in the dashed boxes in B. Reprinted from Methods of Molecular Biology, L. Shi and V. Ladizhansky, Magic angle spinning solid-state NMR experiments for structural characterization of proteins, 895 (2012) 153–165. Reproduced with kind permission from Springer Science and Business Media.

Fig. 3. Representative two-dimensional planes of the 3D CANCO (A) and NCACX (B) experiments collected on the U–13C,15N-labeled ASR. In (B), the NCACX experiment was collected with 50 ms of DARR mixing. In (A), peaks are labeled based on the N[i]–Ca[i] assignments.

The use of simplifying labeling schemes facilitated the completion of the assignment process. We utilized the well-known method of growing cells in media containing alternately labeled [1,3-13C]- or [2-13C]-glycerol as the sole carbon source (1,3-ASR and 2-ASR, respectively). This method creates proteins which are alternately labeled with 13C patterns which do not contain adjacent 13 C labels in most amino acids [29]. This improves the resolution of individual resonances due to the suppression of many one-bond J-couplings, simplifies the overall spectra due to the reduction of the number of resonances, and specifically facilitates the assignment of the aromatic side chains [30,31]. These benefits were all observed in ASR, and experiments preformed on samples grown on alternately labeled glycerols allowed us to extend the assignments to a total of 206 of the 229 residues [32]. The alternate labeling patterns were also crucial for more demanding structural measurements, as discussed below.

3. Structural restraints NMR-based structure determination of proteins is dependent on the ability to obtain a sufficient number of structural restraints. Long-range, e.g., interatomic interhelical distances, and paramagnetic restraints play a special role, as they allow for the definition of the correct protein fold and, under favorable conditions, may provide high-resolution information on the oligomeric interface. Obtaining long-range internuclear restraints in an alpha-helical protein is complicated by a number of factors. First, in uniformly and extensively 13C-labeled proteins weak, structurally constraining dipolar couplings are truncated by strong dipolar couplings from atoms which are closer together [33]. This renders the use of broadband first order recoupling methods inefficient, and necessitates the application of either band-selective techniques, which only recouple the weak and structurally constraining couplings of

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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interest, or second order recoupling methods, which are relatively insensitive to dipolar truncation effects [34,35]. Second, owing to helical geometry, most of the important, long-range, interhelical restraints are obtained between side chain atoms, which have poor dispersion and may be heavily overlapping. Moreover, the unique identification of well-resolved peaks is also problematic and requires the application of iterative structure calculation and cross peak assignment procedures. In our experience, the convergence of such structure calculations is greatly facilitated if the initial structural template is known. We have found that this template can be generated from a relatively small number of unambiguous interhelical distance restraints and knowledge of the secondary structure (discussed below in Section 5). In ASR we used 2-, and 1,3-glycerol labeling combined with second order dipolar recoupling by proton-driven spin diffusion (PDSD) [36], and first order band-selective homogeneously broadened rotational resonance [37] to obtain distance restraints. In 1,3ASR and 2-ASR the elimination of one-bond carbon–carbon dipolar couplings reduced dipolar truncation effects [33]. In Fig. 4A we show examples of 2D PDSD spectra, which display a large number of well-resolved cross peaks. Several of these cross peaks could be assigned unambiguously and were used to initiate a rapidly converging structure calculation procedure, as discussed below. Additional proton–proton contacts were derived from the CHHC experiments [38] which take advantage of the shorter distances between neighboring protons to obtain long-range restraints. Further restraints were obtained through the use of a bandselective recoupling method, homogeneously broadened rotational resonance (HBR2) [37]. HBR2 is a version of rotational resonance (R2) recoupling [39] in which the R2 matching condition is broadened sufficiently enough to simultaneously reintroduce the dipolar interactions between carbonyl atoms and remote aliphatic carbons (Cc, Cd, etc.), while not recoupling the strong one- and two- bond CO–Ca and CO–Cb interactions. The band selective recoupling is achieved by adjusting the spinning frequency to approximately fulfill one of the first order R2 recoupling conditions, (D  nmR, n = 1, 2) and lowering the decoupling power during the R2 mixing

in order to broaden the recoupling condition. Here, D  150 ppm is the approximate chemical shift difference between the CO and Cc and Cd aliphatic resonances, and mR is the spinning frequency. As there are little to no relayed transfer processes, the observed cross peaks correspond to direct interactions and are easily structurally interpretable [37]. To reduce spectral overlap, HBR2 measurements were performed on 2-ASR and 1,3-ASR using a 3D NCOCX pulse sequence with experimental parameters (spinning frequency and the decoupling power) adjusted to establish the CO–Cc polarization transfer. Due to the poor dispersion of the NCO spectrum, the use of threedimensional spectroscopy was necessary and the resolution of the 3D spectra was sufficient to resolve many peaks and assign them with little or no ambiguity. Although the vast majority of the recoupled dipolar interactions between carbonyl and aliphatic side chain atoms were short to medium range and only constrained local structure, eight long-range restraints were observed, including two unambiguous restraints. In Fig. 4A we show a 2D plane of a typical HBR2 spectrum along with a cartoon representation of some of the interhelical interactions recoupled in this experiment. 4. Paramagnetic relaxation enhancements The most commonly measured dipolar 13C–13C and 13C–15N interactions are too weak to report on distances exceeding 5–6 Å. Longer range distance constraints can be obtained through the use of paramagnetic labels, which can be incorporated into a protein through covalent attachment to cysteines that are either natively present or introduced through site-directed mutagenesis. Paramagnetic tags with both anisotropic (e.g., Co2+, most lanthanide ions) and isotropic g-tensors (e.g., nitroxide, Cu2+, Mn2+, etc.) enhance the NMR relaxation rates (paramagnetic relaxation enhancement, PRE) of nearby nuclear spins. The multiple advantages associated with PRE restraints have been extensively discussed in the literature and are used in both solution and solid-state NMR [40,41]. By employing stronger electron-nuclear spin interactions,

Fig. 4. (A) Examples of PDSD, CHHC and HBR2 SSNMR spectra of ASR recorded on sparsely labeled samples (1,3-ASR, 2-ASR, as indicated in the figures). The HBR2 spectrum is a 2D NC plane extracted from the 3D experiment at the carbonyl shift of 178.2 ppm. In all spectra, cross-peaks corresponding to intrahelical restraints (1 < |i  j| < 5) and restraints in the loops connecting adjacent helices (excluding the b-stranded B–C loop) are labeled in blue while interhelical correlations, and correlations defining the short b-hairpin within the B–C loop are labeled in red. Reprinted with modifications from S. Wang, R.A. Munro, L. Shi, I. Kawamura, T. Okitsu, A. Wada, S.Y. Kim, K.-H. Jung, L.S. Brown, V. Ladizhansky, Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein., Nat. Methods, 10 (2013) 1007–1015. (B) Several of the distance restraints used for the initial structural template calculation visualized on the ASR structure. The residues involved in connectivities obtained from the PDSD spectra are shown in blue, while those obtained from HBR2 data are shown in orange. The cytoplasmic side is on top. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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the range of distance restraints accessible to experimental determination is significantly extended. In addition, PRE restraints can be readily assigned, provided spectroscopic assignments for the protein are available. As discussed below in the structure calculation section, these unambiguous PRE restraints can be used to generate a structural template [42], which greatly facilitates the iterative structure calculation process. PREs have also proved to be very useful in the determination of the oligomeric interface. Like many other microbial rhodopsins, ASR forms oligomers in many environments. Numerous biochemical and biophysical evidence pointed to the possibility of oligomerization in ASR. For example, ASR runs as a trimer on a non-denaturing gel, and retains a significant proportion of trimers even when solubilized in SDS [43]. Visible range circular dichroism spectra of ASR solubilized in detergent and reconstituted in lipids both showed bilobe shapes [43] resembling that displayed by the trimers formed by BR [44]. The overall spectral complexity, degeneracy of the NMR spectra, and short-range order of the readily accessible 13C–13C and 1H–1H distance restraints make it difficult to extract internuclear proximities on the interface. However, PREs are ideally suited for this purpose. As the three native cysteines of ASR were found to be inaccessible to paramagnetic reagent binding, it is possible to selectively label cysteines introduced through site-directed mutagenesis. We generated four mutants of ASR with cysteines introduced in the vicinity of the putative interface predicted based on BR homology. Of these, two could be labeled with a nitroxide spin label, MTSL ((1-oxyl-2,2,5,5-tetramethyl-D3-pyrroline-3-methyl) methanethiosulfonate), and one mutant, S26C, produced a data set that yielded intermonomer distances [43]. In Fig. 5A we show the 2D NCA correlation spectra of U–13C,15N labeled S26C ASR with incorporated paramagnetic label and with a diamagnetic control (methanethiol) covalently attached at C26. As expected, several peaks are attenuated or disappear completely in the spectra of the paramagnetic sample. Quantitatively similar results were obtained from the comparison of 13C–13C correlation spectra measured on paramagnetic and diamagnetic ASR. Many residues with attenuated signals belonged to the cytoplasmic halves of helices A and B, and to the adjoining A–B loop, which according to the monomer geometry are within 15 Å from the label. However, there were also strongly affected residues in the cytoplasmic halves of helices D and E (Fig. 5B). The attenuation

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observed in these residues cannot be explained through intramonomer interactions, as both helices are too far (>25 Å) from the label according to even the most conservative estimates of the monomer structure (e.g., the label is oriented inward, and helices are tightly packed). Furthermore, as helices C and F are largely unaffected, the modified side chain of C26 must be oriented away from the alpha-helical bundle. Thus, these PRE effects must be observed between monomers, and therefore provide structural restraints for the intermonomer interface. 5. Structure calculations In Fig. 4A we show fragments of a 2D 13C–13C correlation spectrum collected using proton-driven spin diffusion as the carbon– carbon mixing step. Although the spectrum contains many wellresolved cross peaks, most can only be assigned with some, and usually a high, level of ambiguity, i.e., based on the available assignments, a cross-peak can be attributed to more than one interacting spin pair. This ambiguity creates a challenging problem for structure calculation. Although there are a number of recently developed computational approaches for dealing with spectral ambiguity [45], they are very computationally expensive and may not work for a protein the size of ASR (at least in our experience) unless there is a known structural template that can be used to resolve ambiguity and initiate an iterative cross peak assignment process. For the determination of the structure of ASR, we have developed a multi-step method in which unambiguous structural information, obtained from 13C–13C PDSD, CHHC, HBR2, and PRE measurements, is used to generate an initial low-resolution structural template which can then be used to facilitate the iterative structure calculation and cross-peak assignment. An alternative, more general approach, which was not necessary in the case of ASR, would be to conduct additional PRE measurements and generate a structural template from these, as was recently demonstrated by Jaroniec and co-workers [42]. To facilitate the structure calculation process, a few simplifying assumptions are necessary. In the initial step, we are interested in obtaining a low- to medium-resolution structural template that has the orientation and packing of helices correctly defined. To simplify this calculation, torsional restraints derived from TALOS+ and CSI data were used to fix the helices as semi-rigid bodies.

Fig. 5. (A) 800 MHz 2D NCA correlation spectra of paramagnetically labeled S26C ASR (red) and the diamagnetic control (blue) with several of the attenuated residues labeled. Reprinted with permission from the Journal of the American Chemical society, S. Wang, R.A. Munro, S.Y. Kim, K.-H. Jung, L.S. Brown, V, Ladizhansky, ‘‘Paramagnetic Relaxation Enhancement Reveals Oligomerization Interface of a Membrane Protein’’, 134 (2012) 16995–16998. Copyright 2012, Journal of the American Chemical Society. (B) ASR structure viewed from the cytoplasmic side with nitroxide label (yellow) and side chains of affected residues (green) shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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Although structure calculation in the initial step resulted in a template of low resolution (Fig. 6A), as expected from the small number of restraints, the positions of helices are sufficiently defined, with a backbone root mean square deviation (rmsd) of 3.2 Å. This allows an iterative cross-peak assignment/structure calculation procedure to be initiated. To ensure further rapid convergence of the iterative procedure one can further take advantage of helical geometry, which dictates that any cross peak assigned between residues i and j with |i  j| P 5 must correspond to interhelical interactions. Thus, in the first step, all cross peaks that could be explained by

interactions between atoms in residues i and j with |i  j| < 5 were excluded from consideration, leaving us to deal with only the longrange interhelical cross peaks (|i  j| P 5). The structural template obtained from the initial calculation was used to iteratively resolve the ambiguity of these long-range cross peaks. A total of 79 unambiguous long-range distance restraints could be obtained through this method in the first step. Through the addition of these peaks to the structure calculation, an ensemble of structures with a reduced rmsd of 1.3 Å could be obtained. The calculation was repeated with more stringent violation tolerance, and an additional 100 unambiguous long-range constraints were identified, for a

Fig. 6. Schematic representation of the iterative long-range cross-peak assignment procedure and convergence of the structure calculation. In A, C, and E we show helical wheel representations of ASR viewed from the periplasmic side. The disambiguated long-range contacts used in each step of the structure calculation protocol are depicted by dashed red lines, thin blue solid lines represent restraints between the retinal (shown as blue bar) and the protein, and dashed blue lines represent the intramonomer PRE restraints. In B, D, and F we show ensembles of 10 lowest-energy structures (out of 100) obtained with each set of restraints. The retinal is in yellow, and rms. deviations are calculated for the backbone atoms of a-helices. Reprinted from S. Wang, R.A. Munro, L. Shi, I. Kawamura, T. Okitsu, A. Wada, S.Y. Kim, K.-H. Jung, L.S. Brown, V. Ladizhansky, Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein, Nat. Methods, 10 (2013) 1007–1015. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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total of 211 long-range interhelical restraints. With these restraints added to the structure calculation, the backbone rmsd was reduced to 0.9 Å (Fig. 6). Following this step, the intrahelical cross-peaks were manually analyzed, and resulted in hundreds of short- and medium-range restraints. A final structure calculation utilizing the complete set of restraints yielded an ensemble of 10 structures with a backbone rmsd of 0.6 Å. With the monomer structure determined and the oligomerization interface known from the PRE data, it is possible to identify additional internuclear inter-monomer contacts. Five such contacts could be obtained from the PDSD spectra, which occur between helices B of one monomer and helices D and E of the adjacent monomer. The final structure calculation, which accounted for the oligomerization state (trimer), and both intra- and inter monomer restraints, resulted in an ensemble of 10 structures with a rmsd of 0.8 Å for backbone atoms in the a-helices.

6. Conformational dynamics in ASR The light-modulated interaction of ASR with its transducer implies the ability of the protein to undergo large conformational changes. In the course of structural measurements on ASR we have accumulated sufficient evidence of the presence of dynamic motions in the protein, which may be linked to the proposed biological function. For example, examination of Fig. 7 shows that the extramembranous loop regions, in general, have higher rmsd, are less structurally defined, and are likely mobile. Hydrogen–deuterium (H/D) exchange studies show a high level of accessibility of the exposed loops, and suggest weaker hydrogen bonding for the backbone atoms in these fragments, as well as an asymmetric position of the protein in the bilayer, with the cytoplasmic side being more exposed [11]. Reduced NMR cross-peak intensities of the residues in the cytoplasmic loops further suggest their potential flexibility [46], whereas H/D measurements under illumination show that the light-induced conformational changes in ASR produce intermediate states which differ in the water accessibility of the cytoplasmic side [47]. In addition to these largely indirect indicators of dynamics in ASR, SSNMR offers ways to directly probe both the amplitudes and time scales of motions. In particular, dipolar order parameters report on the amplitudes of motions occurring on the submicrosecond time scale and the nuclear spin relaxation rates report on the time scale of motions. Longitudinal relaxation is sensitive to fast

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picosecond-nanosecond motions, and transverse relaxation can be used to characterize the time scale of slower, nanosecond– microsecond motions. While dipolar order parameter measurements have found widespread use in SSNMR [48,49], the effective transverse relaxation rates are usually dominated by the coherent effects of non-averaged dipolar couplings. Recently, two approaches have been devised to overcome this problem. In the first, extensive deuteration, followed by backexchange, is used to dilute the proton bath, reduce interproton couplings, and diminish the coherent effects on the relaxation pathways [50]. Alternatively, it has been shown that this problem can also be surmounted under fast magic angle spinning conditions. Proton-driven spin diffusion contributions to longitudinal relaxation rates become negligible above 20 kHz and 60 kHz for 15 N and 13C atoms, respectively [51]. Likewise, fast MAS rates (>45 kHz), in combination with a spinlock, result in long 15N R1q relaxation times dominated by the stochastic contribution to the coherence lifetimes, and can therefore be used to study motions on the nanosecondmicrosecond time scale [52]. To characterize the amplitude of motions in ASR we measured four dipolar couplings per residue, 15N1HN, 13Ca1Ha, 13C0 15N, and 13Ca15N. Surprisingly, we observed little variation in the order parameters between the transmembrane and solvent exposed regions, suggesting that these domains undergo motions of similar amplitudes and, overall, are rigid. In Fig. 8A and B we show examples of 15N–1H and 13Ca–1Ha order parameters. The site-specific R1q transverse relaxation rates shown in Fig. 8C revealed a very different picture, showing significant variations within helices and especially between helices and the extracellular B–C and F–G loops. Elevated relaxation rates for these loops indicate a significantly larger contribution from slower motions, which were estimated to have a timescale of tens of nanoseconds for the helices, and of up to a hundred nanoseconds for the loops (Fig. 8D). Both the transmembrane helices and the B–C loop represent well-defined structural elements stabilized by intrahelical or interstrand hydrogen bonds. Similarly, the F–G loop, although less structurally defined, shows a significant amount of b-structure, and contains residues whose amides are protected from exchange with water [28]. This, and the overall slow times scales of motions estimated for the protein, suggest the possibility of collective motions. To investigate if the collective motions model could adequately explain the experimental data, we used the 3D Gaussian axial fluctuation (3D GAF) method [53] to model the seven helices and the B–C and F–G loops as rigid bodies moving with a single

Fig. 7. (A) 10 lowest energy structures of the ASR monomer obtained from the final trimer structure calculation. (B) The lowest energy trimer structure from the cytoplasmic side. In both figures the retinal is shown in yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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Fig. 8. Site-specific (A) 1H15N and (B) 1Ha13Ca one-bond order parameters, (C) 15N R1q relaxation rate constants, and (D) motional correlation times estimated from the 1 H15N order parameters and relaxation rates with arrows indicating cases where only the lower bound could be extracted. Red lines in (A) and (B) correspond to the rigid limit. Error bars define a 95% confidence level interval and the secondary structure is displayed on top of the graphs. Modified with permission from the Journal of the American Chemical society, D.B. Good, S. Wang, M.E. Ward, J. Struppe, L.S. Brown, J.R. Lewandowski, V. Ladizhansky, Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR, 136 (2014) 2833–2842. Copyright 2014, Journal of the American Chemical Society.

Fig. 9. Time scales, amplitudes, and directions of collective motions in ASR estimated using 3D GAF analysis of the transverse relaxation rates R1q and backbone order parameters S2NH ; S2Ca Ha . (A) Cytoplasmic side view, (B) extracellular side view, (C) side view. Reproduced with permission from the Journal of the American Chemical society, D.B. Good, S. Wang, M.E. Ward, J. Struppe, L.S. Brown, J.R. Lewandowski, V. Ladizhansky, Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR, 136 (2014) 2833–2842. Copyright 2014, Journal of the American Chemical Society.

time constant. The resulting motional model is depicted in Fig. 9. Although this approach gives the correct time scales, similar to those obtained using the model-free approach (Fig. 8D), it likely overestimates the amplitudes. Undoubtedly, the 3D GAF modeling was based on a data set which was intrinsically insensitive to fast picosecond time scale motions, and although such motions contribute negligibly to the transverse relaxation rates, they may have a profound effect on the order parameters. Thus, the amplitudes of motions shown in Fig. 9 are likely overestimated, and should be taken only as indicators that slower collective motions may be present in helices. However, the elevated relaxation rates for the B–C and F–G loops indicate with certainty that these regions undergo slow motions. Unfortunately, we lack, at present, the experimental relaxation data for residues on the cytoplasmic side (Fig. 8C), which prevents us from drawing direct conclusions about the time scale of motions affecting these residues. 7. Summary and conclusions Here, we have discussed the essential solid-state NMR methodologies for structure determination and the characterization of the

dynamics of polytopic a-helical membrane proteins using Anabaena Sensory Rhodopsin (ASR) as an example. The main advantage of solid-state NMR for these studies is that membrane proteins can be retained in the native-like lipid environment. Although the preparation of samples requires some optimization of reconstitution conditions, e.g., protein-to-lipid ratio and the type of lipids, it is generally straightforward and a breadth of biochemical conditions can be applied to study protein function with only minor modifications to sample preparation protocols. There are several examples of membrane proteins of different classes which yield solid-state NMR spectra of excellent resolution, which facilitates both structural and dynamical analysis [25]. For ASR, the high spectral resolution allowed for extensive spectroscopic assignments to be obtained from three samples with complementary labeling schemes. The structure of ASR and its oligomeric state were determined using a set of internuclear distances and PRE restraints which provide information on both the backbone and side chain conformations. Furthermore, recent advances in fast MAS probe technology have enabled the characterization of protein dynamics using relaxation measurements. The application of these methods to ASR has suggested the presence of collective

Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017

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motions in some of the exposed, ordered loops and also in the transmembrane helices. Although sensitivity remains the main limiting factor in studies of large membrane proteins, it will likely be improved in the future due to rapidly developing proton detection methods [16]. Thus, we anticipate that the existing and new, emerging methodologies will set the stage for solid-state NMR to become the primary method for studying membrane proteins.

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Please cite this article in press as: M.E. Ward et al., Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin, J. Magn. Reson. (2015), http://dx.doi.org/10.1016/j.jmr.2014.11.017