Proton-Detected NMR Spectroscopy of Nanodisc-Embedded

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Proton-Detected NMR Spectroscopy of Nanodisc-Embedded Membrane Proteins: MAS Solid-State vs. Solution-State Methods Nils-Alexander Lakomek, Lukas Frey, Stefan Bibow, Anja Böckmann, Roland Riek, and Beat H Meier J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06944 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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The Journal of Physical Chemistry

Proton-Detected NMR Spectroscopy of Nanodisc-Embedded Membrane Proteins: MAS Solid-State vs. Solution-State Methods Nils-Alexander Lakomek1 ‡, Lukas Frey1 ‡, Stefan Bibow1 ‡, Anja Böckmann2*, Roland Riek1*, Beat H. Meier1* 1

ETH Zürich, Physical Chemistry, Vladimir-Prelog-Weg 2, 8093 Zürich, Switzerland

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Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367 Lyon, France

ABSTRACT: The structural and dynamical characterization of membrane proteins in a lipid bilayer at physiological pH and temperature and free of crystal constraints is crucial for the elucidation of a structure/dynamics – activity relationship. Towards this aim, we explore here the properties of the outer-membrane protein OmpX embedded in lipid bilayer nanodiscs using proton-detected magic angle spinning (MAS) solid-state NMR at 60 and 110 kHz. [1H,15N]correlation spectra overlay well with the corresponding solution-state NMR spectra. Line widths as well as line intensities in solid and solution both depend critically on the sample temperature, and, in particular, on the crossing of the lipid phase-transition temperature. 110 kHz MAS experiments yield well resolved NMR spectra also for fully protonated OmpX and both below and above the lipid phase transition temperature.

The determination of the 3D atomic-resolution structure and the dynamics of membrane proteins embedded in and interacting with a lipid bilayer membrane are essential for a detailed understanding of membraneprotein functions. These interactions are influenced by the properties of lipids, such as the degree of saturation, chain length, chain size, lipid head groups charge and polarity.1 An apparent physical property of lipid bilayers is the temperature-dependent change in fluidity at the phase transition. Below the lipid phase-transition temperature the bilayer is in a “solid-like” gel phase, without significant translational motion of lipids or protein.2 Above the lipid phase-transition temperature the lipid bilayer is in a “liquid-like” phase with substantial lateral diffusion.2-3

like lipid environments, such as lipid bilayer nanodiscs.5 However, both methods provide only static snapshots at cryogenic temperatures, while for a detailed and comprehensive understanding of the structure/dynamicsfunction relationship of membrane proteins, the dynamics of membrane proteins embedded in and interacting with a dynamic lipid bilayer needs to be taken into consideration. The dynamics of membrane proteins and the interplay between lipid membranes and membrane proteins have been investigated by various biophysical techniques at physiological temperatures, including Electron Paramagnetic Resonance (EPR) spectroscopy,6 Fluorescence Spectroscopy7 and NMR Spectroscopy,8 but high-resolution studies providing detailed residue-specific dynamical insights around the phase transition of lipid bilayers have been rare so far.9

X-ray crystallography is currently the most successful method for high-resolution structure determination of membrane proteins, frequently embedded in detergents or in lipid cubic phases.4 Recent technological advances in cryo-electron microscopy promise to ease the requirements for sample preparation by avoiding crystallization, allowing thereby the use of more native-

Both solution- and solid-state NMR studies have investigated membrane protein structure and dynamics at temperatures close to physiological temperature. The two techniques typically require different sample conditions. For solution-state NMR to work, the overall tumbling (where anisotropic interactions will be averaged) of the

Introduction

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entire membrane protein complex (including the lipid environment), needs to be at least on the tens-of-ns time scale or faster. Therefore, the overall tumbling limitation restricts solution-state NMR investigations (of the protein backbone) to an overall molecular weight below ca 100200 kDa.10-11 Hence, membrane proteins are usually reconstituted in small-sized detergent micelles, bicelles or amphipols but not in liposomes.8, 12-13 In contrast, solidstate NMR applies if the overall motion is arrested, e.g. by sedimentation of the system, which demands a certain size for the lipid-protein assembly. Membrane proteins for MAS solid-state NMR investigations are therefore usually reconstituted in 2D lipid crystals or in liposomes, and because of severe line-broadening in the protondimension (due to proton-dipolar interactions), usually detection on the 13C nucleus is applied.14-21 Limitations arise from relaxation effects, either caused by extrinsic or intrinsic protein motions and by the complexity of the spectrum for large systems. To enable proton detection and allow for sufficient resolution also in the protondimension, either high deuteration levels22-23 or fast magic-angle spinning >40 kHz are required.24-27 For fully protonated samples magic-angle spinning faster than 100 kHz is of advantage in order to sufficiently average out the anisotropic interactions.28-30 However, there have been fewer studies on membrane proteins embedded in 2D-lipid crystals31-32 or liposomes / lipoparticles 33-35 where fast MAS (> 40 KHz) and proton-detection are applied.

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Experimental Section Sample preparation Outer membrane protein OmpX expressed in E. coli in inclusion bodies was isotopically labeled with 2H,13C, and 15 N (as well as 15N only); amides were protonated to either 75% or 100% by back-exchange as described in detail in the Supporting Information.38, 47, 50 The protein was reconstituted in n-dodecylphosphocholine (DPC, FOS-12, Avanti Polar Lipids, Inc.) and then assembled into membrane scaffolding protein (MSPΔH5) nanodiscs containing deuterated 1,2-dimyristoyl-sn-glycero-3phosphocholine (14:0 PC, DMPC) / 1,2-dimyristoyl-snglycero-3-phospho-(1'-rac-glycerol) (14:0 PG, DMPG) (ratio 3:1) lipids, as described in detail by Hagn et al.38 and Frey et al.47. For all NMR experiments a 20mM Tris, 100 mM NaCl, pH 7.4 buffer was used. These nanodiscs were concentrated to 268 µM (233 µM) for solution-state NMR measurements, or centrifuged into a 0.7 mm or 1.3 mm Bruker solid-state NMR rotor51-52 at 49 400 g for 16 hours for solid-state NMR measurements. The total amount of OmpX in the solution state NMR sample was thus 0.7 mg (0.6 mg), while for the solid-state NMR measurements (see below) the 1.3 mm rotor contained 0.65 mg of OmpX, and for measurements using an 0.7 mm rotor, the sample volume was 0.14 mg (0.12 mg), respectively. This allows a direct comparison between solid-state and solution-state NMR measurements on a very similar sample. For a detailed description of expression and purification of MSPΔH5 and OmpX, reconstitution of OmpX into lipid nanodiscs (NDs), filling of 0.7mm rotors and 1.3 mm rotors as well as an estimation of the lipid-to protein ratio we refer to the Supporting Information.

The recently developed lipid-bilayer nanodisc technology36-49 has the potential to allow for solid- and solution NMR experiments on essentially the same sample. This may allow for a combined investigation of membrane protein structures and dynamics by both techniques and also allows investigation by cryo-EM on the same sample.5 Here, we study temperature-induced lipid-mediated effects on E. coli’s outer membrane protein X (OmpX) embedded in lipid nanodisc using proton-detected solidstate NMR on ∼0.15 mg of membrane protein. A strong temperature dependence is observed, with highest signal intensities at temperatures below the lipid phase transition, but highest spectral resolution above the lipid phase transition. Varying the lipid chain lengths from DMPC/DMPG (14 C-atoms, Tm = 24 °C) to DPPC (16 Catoms, Tm = 41 °C) caused an off-set in the observed temperature dependence, consistent with the higher lipid phase transition temperature of DPPC. In solution-state NMR highest spectral resolution is found above the phase transition temperature of the lipids. We show that reconstitution of a membrane protein in nanodiscs allows for the study of membrane proteins by both solution- and solid-state NMR, allowing for a characterization of the structure and dynamics above and below the lipid phase transition temperature.

NMR spectroscopy Data acquisition

Solid-state NMR experiments Solid-state NMR experiments at 110 kHz MAS were performed on a Bruker AVANCE III 850 MHz spectrometer using a 0.7 mm triple resonance MAS probe (Bruker Biospin). The gas flow for sample cooling was set to 200 l/h without further cooling for the maximal temperature and 480 l/h with maximal cooling for the minimal temperature. Spectra were referenced to DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid), the experimental temperature was determined via the chemical shift, δ, of the up-field component of the waterline in a 1H 1D experiment using the formula T [°C] = 455

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K- 90δ.51 Experimental temperatures were ca. 37 °C ± 5 °C (deuterated sample) or 36 °C ± 5 °C (protonated sample) (for the high temperature experiment) and 20 °C ± 5 °C (for the low temperature experiment), as calibrated on the up-field water-line in a 1H 1D experiment.51 (Limitations in signal to noise and the shape of the water line did not allow a more accurate temperature calibration.) 2D [1H,15N]-correlation spectra were recorded using the NMR experiment shown in Figure S1. Experimental parameters are summarized in Supplementary Table S1.

Spectra were zero-filled to 2k real points in the direct and 1 k real points in the indirect dimension. The solid-state spectrum recorded on 2H,13C,15N-labeled OmpX (75% of amides protonated) at 110 kHz MAS and the corresponding solution-state TROSY spectrum (Fig. 1) were processed using the NMR Pipe software package.54 For the direct (1H) dimension, a shifted sine-bell window function was applied on the acquired free induction decay (FID), using a shift of 0.4 π = 72° and a factor 3 in the exponent; the window function ended at 0.95 times the acquisition time. Then the FID was zero-filled up to 4k points and a fast Fourier transformation was performed. For the indirect dimension the same window function and parameters were applied. Spectra were zero-filled up to 2k points before Fourier transformation. Both solidstate and solution-state spectra were processed the same way, in addition automatic polynomial baseline correction (zero order) was applied for the solution-state spectrum in the direct dimension.

Temperature dependent solid-state NMR experiments at 60 kHz MAS were recorded on a Bruker AVANCE III 850 MHz spectrometer using a 1.3 mm triple resonance MAS probe (Bruker Biospin). Spectra were referenced to DSS. The gas flow for sample cooling was varied between 1500 l/h and maximal cooling for the minimal temperature and 200 l/h without further cooling but gentle heating for the maximal temperature. The first increment of a 2D [1H,15N]-correlation experiment was recorded (for experimental details compare Supplementary Table S1).

Spectra were plotted using the Analysis CCPN software package.55 All spectra were plotted using 20 contour levels and a level multiplier of 1.2, except for the spectra processed using NmrPipe (Fig. 1), where 32 contour levels and a multiplier of 1.1 were used. Plotting parameters were identical for the corresponding solution-state TROSY spectra.

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N and 1H coherence life times were measured using a spin-echo delay, by introducing a spin-echo either directly prior to t1 evolution for the 15N coherence life time measurement or prior to 1H detection for the 1H coherence life time measurement. The spin-echo based experiments were recorded in an inter-leaved fashion with two different relaxation delays of Δ = 4 ms and Δ = 1 ms for the reference experiment. The experimental time for each 2D plane was 4 h (64 transients for each of the 128 increments), resulting in a total experimental time of 8 h for each relaxation experiment.

Transfer of spectral backbone assignments 2

H,13C,15N-labeled OmpX (100% of amides protonated) in DMPC/DMPG nanodiscs was assigned previously by Hagn et al. (BMRB entry 18796.str).38 The backbone assignment was transferred to the solution-state NMR spectrum and further to the solid-state NMR spectrum as described in detail in the Supporting Information.

Solution-state NMR experiments Spectra were recorded on Bruker Avance III 600 MHz, 700 MHz and 900 MHz spectrometers, all equipped with cryogenic probes, the experimental temperatures were 45 °C, 43 °C, 36 °C, 30 °C and 20 °C. [1H,15N]- TROSY experiments53 were recorded using experimental parameters summarized in Supplementary Table S2.

Spectra analysis Spectra shown in Figure 1 were analyzed in terms of peak positions, line widths, intensities and volumes using dedicated NMR Pipe scripts (http://www.nmrscience.com/),54 provided by Dr. Frank Delaglio (Institute for Bioscience and Biotechnology Research, National Institute of Standards, Maryland, Rockville, Maryland, USA). For the line width analysis, peaks were fitted with a Lorentzian line shape.

Spectral processing Solid-state spectra recorded at 60 kHz MAS or on fully protonated OmpX at 110 kHz MAS (Fig.2 to 3 and Fig. 4) were processed using the software Bruker Topspin 3.5. Spectra were zero-filled to 4k real points in the direct and 1k real points in the indirect dimension. A shifted sinebell window function (SSB=3) was applied and the effective acquisition duration was limited to 1k points in the direct dimension, resulting in an effective acquisition time of 15 ms in the direct dimension. For the corresponding solution-state TROSY spectra the same window function (shifted sine-bell, SSB=3) was used.

Results and Discussion OmpX was expressed in E.coli inclusion bodies and isotopically labeled with 15N, and for some samples also with 13C and 2H, and was then reconstituted into DMPC/DMPG lipid nanodiscs with a diameter of about 8 nm. These OmpX nanodiscs were either studied by

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spectrum (compare Supporting Information Table S3 and Figure 1B). While non-assigned residues (due to overlap) are distributed across the entire protein, the majority of residues which are invisible in the solid-state spectrum, are located in the L5 loop region (residues 91-103) and the adjacent N-terminus of β-strand 6 (residues 104-115) or the L6 loop (residues 116-121) (compare Figure 1B and Table S3). Those loop regions belong to the solventexposed part of OmpX and showed dynamics both on the fast (ps-ns) and slow (µs-ms) time scale, as indicated by chemical-exchange (Rex) contributions in a recent solution-state NMR study on OmpX in nanodiscs.47

solution-state NMR at a concentration of about 250 µM, or centrifuged into a 0.7 mm or 1.3 mm Bruker solid-state NMR rotor45 at 49 400 g for 16 hours (see Experimental Section and Supporting Information) and then studied by proton-detected solid-state NMR methods.

Both solution- and solid-state NMR yield resolved protondetected spectra of 2H,13C,15N- OmpX embedded in lipid DMPC/DMPG nanodiscs with comparable NMR fingerprints. Two-dimensional [1H,15N]-correlation spectra of 2H,13C,15Nlabeled OmpX in lipid DMPC/DMPG nanodiscs were recorded at a magic-angle spinning (MAS) frequency of 110 kHz and at a temperature of 37 °C ± 5 °C. The spectra were compared to solution-state [1H,15N]-TROSY spectra recorded at 45 °C (the higher temperature was selected to optimize spectral quality, i.e. high signal to noise and high chemical shift resolution) (Figure 1). The solutionstate spectrum has been reported and assigned previously.38 Cross-peak positions of solution- and solidstate spectra match closely and yield a very similar “fingerprint” of the protein, indicating that OmpX is in the same conformation in both samples. We conclude that the high g forces experienced during MAS (up to 12 million g) apparently did not impact the integrity of the sample. The resolution of solid-state spectra starts approaching the one for solution-state (Figure 1), however there is still significant need for improvement.

Solid and solution spectra of protonated and deuterated OmpX recorded above the lipid phase transition Deuteration of proteins and subsequent back-exchange of protons is spectroscopically a great advantage, as it reduces proton relaxation and prolongs 15N and 1H coherence life times. Therefore deuteration enables highresolution in solution-state TROSY-spectroscopy of large biomolecules,10 but also allows for high-resolution spectra in solid-state NMR (see above). However, deuteration may lead to a significant loss of expression levels which may get detrimental because of the frequently low yield of membrane proteins under standard conditions. A further disadvantage of deuteration is that highly protected 2H15 N moieties, which are frequently present in transmembrane regions, are difficult to re-protonate and thus escape detection in 1H experiments, unless protein refolding is established, which is often not straight forward. Alternatively, deuterium-detection based methods56 or recent alternative deuteration schemes34 may be employed.

In the solid-state spectrum, 134 cross peaks out of the 143 possible ones can be picked, of which 90 could be tentatively assigned by transferring the solution-state NMR assignment. The two spectra show only small 1H chemical-shift differences of less than 0.1 ppm (median 0.02 ppm, Figure S2A) and 15N chemical shift differences of less than 1 ppm (median 0.16 ppm, Figure S2B). The 1H line widths in the solid-state spectrum vary between 40 and 80 Hz for most resonances, compared to line widths between 30 and 40 Hz for most resonances in the solution-state [1H,15N]-TROSY spectrum (Figures S2C and E). The 15N line widths in the solid-state spectrum vary between 50 and 90 Hz for most resonances, compared to line widths between 25 and 35 Hz for most resonances in the solution-state [1H,15N]-TROSY spectrum (Figures S2D and F).

Therefore, we tested whether cross-polarization(CP)based 2D [1H,15N]-correlation experiments on fully protonated 15N-labeled OmpX embedded in lipid DMPC/DMPG nanodiscs recorded at 850 MHz and at a MAS frequency of 110 kHz and at a temperature of 36 ± 5 °C still yield well-resolved 2D [1H,15N]-correlation spectra. The corresponding spectrum is shown in Figure 2B. While the linewidth is broadened by a factor 2 in the proton dimension compared to the deuterated sample (compare Figure 2A and 2B), the spectrum still shows resolved peaks (Figure 2B). The corresponding TROSY spectrum of protonated 15N-labeled OmpX is of significant lower intensity, as expected, however, surprisingly, spectrally still well resolved (Figure 2D, see Figure 2C for spectrum on deuterated OmpX). In addition, considerable broad signals show up in the random-coil region of the spectrum which are of unknown origin (Figure 2D). Their origin is however not in sample degradation, as the changes are reversible (see Figures S3 and S4).

Due to the difference in line widths, several resonances remain unresolved in the solid-state NMR spectrum but can be clearly separated in the solution-state spectrum. We discriminate between residues which cannot be assigned in the solid-state spectrum due to spectral overlap and those which remain invisible in the solidstate spectrum but are observable in the solution-state

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Figure 1. Superposition of the solid-state and solution-state NMR [1H,15N]-correlation spectra of 2H,13C,15N-labeled OmpX in DMPC/ DMPG lipid nanodiscs highlights their resemblance. A) The black contours are the solid-state spectrum of ca. 0.14 mg 2H,13C,15N-labeled OmpX (sample A1, compare Table 1) at 37 ± 5 °C, the red contours the solution NMR spectrum of 268 µM (equivalent to ca. 0.7 mg in the NMR active volume) 2H,13C,15N-labeled OmpX at 45 °C (sample A2). The assignment was taken from38. The side-chain amide signals of Gln, due to a shorter (transfer time) value for the INEPT transfer in the [1H,15N]- TROSY spectrum, are labeled by an asterix. B) Two different views of the solution-state NMR structure of OmpX (2M06),38 with residues invisible in the solid-state spectrum but visible in the solution-state spectrum colored in red and residues well resolved in the solution-state spectrum but unassigned in the solid-state spectrum due to peak overlap colored in blue.

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Acronym

A1

sample

2

A2 13

15

H C N

NH 75%

2

13

15

H C N

B1

B2

C

15

15

2

N

N

NH 100%

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13

15

H C N

NH 100%

2

13

D2 15

H C N

NH 100%

2

H13C 15N

NH 100%

lipid

DMPC/DMPG

DMPC/DMPG

DMPC/DMPG

DMPC/DMPG

DMPC/DMPG

DPPC

DPPC

rotor

0.7mm

liquid

0.7mm

liquid

1.3mm

1.3mm

liquid

protein mass

268 µM /

233 µM /

290 µM/

in sample

0.14 mg

0.7mg

0.12 mg

0.6 mg

0.65mg

0.65mg

0.7mg

LPR (w/w)

5:2

5:2

5:2

5:2

5:2

5:2

5:2

1,2,3

1,2,3

2,3

2,3

4

4

4

(OmX/lipid) Figure

Table 1. Summary of OmpX samples shown in the spectra.

OmpX (sample A1, see Table 1), recorded at 850 MHz, 110 kHz MAS and 37 °C ± 5 °C, for a total experimental time of 24 h. (The spectrum is identical to the one shown in Figure 1A.) B) Solid-state spectrum of ca. 0.12 mg 15Nlabeled OmpX (sample B1), 36 °C ± 5 °C, 48 h. C) Solution-state TROSY spectrum of ca. 0.7 mg 2H,13C,15Nlabeled OmpX (sample A2), recorded at 600 MHz using a cryogenic probe and at an experimental temperature of 45 °C. The total experimental time was 8 h. (The spectrum is identical to the one shown in Figure 1A.) D) Solution-state TROSY spectrum of ca. 0.6 mg 15N-labeled OmpX (sample B2), 900 MHz, cryogenic probe, 36 °C, 48 h. E-F) Representative traces of the spectra of 2H,13C,15N-OmpX (panel A and C), solid (black) vs. solution (red). (Intensities were normalized to achieve comparable noise level in both spectra.) G-I). Representative traces of the spectra of 15N-labeled OmpX (panel B and D), solid (black) vs. solution (red).

Solid and solution spectra of protonated and deuterated OmpX recorded below the lipid phase transition A spectral comparison of solution-state and solid-state spectra recorded at 20° C, and thus below the phase transition temperature of 24 °C, is shown in Figure 3, both for deuterated and fully protonated protein. When comparing to the measurements at 36 °C (Figure 2), solidstate spectra at 20° C still show similar resolution (Figure 3A and 3B), while solution-state spectra greatly deteriorate at 20° C attributed to the longer overall tumbling correlation times, both for the deuterated and more strongly for the fully protonated samples (Figure 3C and 3D). As mentioned also for the spectra above the

Figure 2. Comparison of solid and solution-state NMR [1H,15N]-correlation spectra of 15N-labeled and 2H,13C,15Nlabeled OmpX (75% or 100% of amides protonated) embedded in DMPC/DMPG lipid bilayer nanodiscs recorded above the lipid phase transition temperature. A) Solid-state spectrum of ca. 0.14 mg 2H,13C,15N-labeled

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phase transition, these changes are reversible and no degradation of the sample takes place (Figure S3 and S4).

increase with temperatures (Figure S7). Therefore, a transverse relaxation effect does not seem responsible for the broadening at lower temperature but inhomogeneous line broadening seems to be the main effect. This inhomogeneous broadening appears reduced above the lipid phase transition temperature.

To systematically investigate these temperature dependent effects, further experiments on the deuterated sample were performed at a MAS frequency of 60 kHz, using a 1.3 mm Bruker probe which has better variabletemperature capabilities than the 0.7 mm probes (Figure 4). Lower temperatures lead to improved spectral intensity (Figure 4A) in the solid-state. The temperature dependence of the signal integral (Figure 4A) is provided in Figure 4C (blue dots). It monotonously decays as a function of increasing temperature, with indications for a sigmoidal-like function with a midpoint at ca. 25 °C, close to the phase-transition temperature of the lipids (24 °C for DMPC and 23 °C for DMPG, respectively). To monitor the lipid transition, 1H solution state NMR intensities of the lipid aliphatic groups were recorded and plotted against the temperature (Figure 4C, black dots). While there is no discontinuous behavior at the phase-transition point for the solution-state lipid signal (black dots) as well as the solid-state protein signal, both intensities change with a function of temperature reciprocal to each other, suggesting that the lipid dynamics within the nanodisc is (at least partially) responsible for the observed signal intensity change of the protein signals in the solid-state NMR measurements (Figure 4C). To exemplify this finding more clearly, we inverted and normalized the lipid intensities from the solution-state NMR measurements and plotted them as black line (Figure 4C). Indeed, those inverted lipid intensities empirically follow a similar trend as the solid-state protein signal intensities (blue dots). A temperaturedependent change of lipid fluidity in DMPC lipid nanodiscs and DMPC liposomes has recently been studied by 2H solid-state NMR, with the phase transition found to be more gradual in lipid bilayer nanodiscs than in liposomes. Below the lipid phase transition temperature, lipids were more mobile in nanodiscs than in liposomes.57 The membrane protein proteorhodopsin on the other hand showed overall increased order in DMPC lipid nanodiscs compared to liposomes of the same lipid.58

Figure 3. Side-by side comparison of solid and solutionstate NMR [1H,15N]-correlation spectra of 15N-labeled and 2 H,13C,15N-labeled OmpX (75% or 100% of amides protonated) embedded in DMPC/DMPG lipid bilayer nanodiscs, recorded below the lipid phase transition temperature. A) Solid-state spectrum of ca. 0.14 mg 2 H,13C,15N-labeled OmpX (sample A1, recorded at 850 MHz, 110 kHz MAS, 20 °C ± 5 °C, total experimental time 24 h). B) Solid-state spectrum of ca. 0.12 mg 15N-labeled OmpX (sample B1, 850 MHz, 110 kHz MAS, 20 °C ± 5 °C, total experimental time 24 h). C) Solution-state TROSY spectrum of ca. 0.7 mg 2H,13C,15N-labeled OmpX (sample A2, 600 MHz, cryogenic probe, 20 °C, total experimental time 4 h). D) Solution-state TROSY spectrum of ca. 0.6 mg in 15N-labeled OmpX (sample B2, 700 MHz, cryogenic probe, 20 °C, 8 h).

Crossing the lipid phase transition: effect on solid state NMR spectra While the solution-state spectra improve with increasing temperature in intensity and resolution, as result of faster overall tumbling (Figure S5), the situation is different for the solid-state NMR spectra. Below the lipid phase transition temperature, higher intensities are observed which go along with broader lines, while above the phasetransition temperature lower signal intensities with sharper lines are observed (compare Figure 3A and Figure 1A, Figure S6). A rough estimate of 1H and 15N R2’ coherence decay rates show that both rate constants

7



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To corroborate our finding of a strong influence of the lipid phase transition onto the spectral quality, 2H,13C,15Nlabeled OmpX was embedded in nanodiscs comprising a different lipid with a higher lipid phase-transition temperature. We used non-deuterated 1,2-dipalmitoyl-snglycero-3-phosphocholine, 16:0 PC (DPPC) with a lipid transition temperature of 41 °C. Again, a temperature scan of the first increment of the 2D experiment (Figure 4B) was collected. Indeed, similarly to the finding with the DMPC/DMPG-containing nanodiscs, decreasing intensities are observed for increasing temperatures, and again a reciprocal temperature-dependent change of the protein signal intensities in the solid-state NMR and the solution-state lipid signal intensities (Figure 4D). The signal intensity changes are however off-set to higher temperatures, proportionally to the difference in the lipid phase transition temperature (i.e. 41 °C vs. 24 °C, Figures 4B and 4D). These findings indicate that the lipid dynamics indeed influences the overall body motions of the membrane protein under study (e.g. rotational diffusion of the membrane protein in the lipid fluid phase3, 40, 57-61). These dynamics can average the 15N-1H dipolar coupling and therefore scale down the CP-transfer efficiency, leading to concomitant decrease of signal intensity of the cross peaks. The dynamics apparently occur on faster rather than on a slower (T1ρ) timescale, as the temperature-dependent decrease of CP-transfer efficiency cannot be explained by a T1ρ relaxation effect, since the latter shows only weak temperature dependence (Figure S8).

Solid- and solution-state NMR of membrane proteins embedded in nanodiscs allow the analysis of the structural and dynamical effects of the lipid phase transition

Figure 4. 1D [1H,15N]-correlation solid-state NMR spectra of OmpX in lipid nanodiscs at various temperatures show that highest spectral intensities are obtained below the phase transition temperature. A) Detailed temperature dependence of the first increment of CP-based protondetected solid-state NMR [1H,15N]-correlation spectra of 2 H,13C,15N-labeled OmpX (100% of amides protonated) embedded in lipid DMPC/DMPG nanodiscs (sample C), recorded at a MAS frequency of 60 kHz at a magnetic field of 850 MHz 1H Larmor frequency. (B) Same as A) but for OmpX in DPPC nanodiscs (samples D1, D2). (C) Normalized integrated bulk intensities of the NMR spectra depicted in (A) are plotted against the temperature (blue dots). Normalized integrated bulk intensities of 1H detected solution-state NMR spectra of DMPC/DMPG lipids (sample A2) monitor the headgroup dynamics around the lipid phase transition (black dots). The inverted and normalized lipid intensities are plotted as black line. The phase-transition temperature (24 °C) is indicated by a vertical black line. (D) Same as A) but for OmpX in DPPC nanodiscs (samples D1, D2). The phase transition temperature of DPPC (41 °C) is indicated by a vertical black line (D).

The lipid phase transition is considered to play important roles in membrane protein function and its regulation.62-63 Our data indicate that phase transition-induced structural and dynamical changes of a membrane protein embedded in a lipid bilayer nanodisc can be followed residue specifically by employing both solid-state as well as solution-state NMR. To cover the phase transition thoroughly it is suggested to combine the two methods. Within this context it is also noteworthy that both methods require only little protein quantities, with the presented solid-state NMR sample containing only ca. 0.15 mg of the membrane protein under study. In addition, both solid and even solution-state NMR yield quite well resolved spectra of a protonated 15N-labeled membrane protein preparation. Because the yields of membrane protein purifications are usually small and may further drop significantly upon deuteration, the ability to work

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The Journal of Physical Chemistry Acknowledgements

with protonated protein can be an important prerequisite in many applications.

This work was supported by the EU Marie-Curie program (grant number 627767, to N.L.), the ETH Zurich, the Swiss National Science Foundation (Grant 200020_159707 and 200020_146757), and the French ANR (ANR-12-BS08- 0013-01, ANR-14-CE09-0024B).

Conclusion

Resolved proton-detected CP-based 2D [1H,15N]correlation spectra on sub-milligram (< 0.15 mg) quantities of lipid bilayer nanodisc embedded OmpX were recorded using solid-state NMR with fast MAS (110 kHz), both for deuterated and for fully protonated samples. The resonance positions overlay well with a [1H,15N]- TROSY solution-state NMR spectrum of OmpX in the same nanodiscs, indicating that nanodiscs are stable under ultracentrifugation at up to 12’000’000 g in the NMR rotors. Also in the absence of deuteration, solidstate NMR with fast MAS (here 110 kHz) allows the study of membrane proteins difficult to deuterate. The presented approach shall therefore allow further investigation of the relationship between membrane protein dynamics and lipid bilayer status when crossing the lipid phase transition, considered to be pivotal in the regulation of membrane protein function. 62-63

REFERENCES (1) van Meer, G.; Voelker, D. R.; Feigenson, G. W., Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Bio. 2008, 9, 112-124. (2)Chapman, D.; Urbina, J., Biomembrane phase transitions. studies of lipid-water systems using differential scanning calorimetry. J. Biol. Chem. 1974, 249, 2512-2521. (3) Peters, R.; Cherry, R. J., Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers: experimental test of the Saffman-Delbruck equations. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 4317-4321. (4) Bill, R. M.; Henderson, P. J. F.; Iwata, S.; Kunji, E. R. S.; Michel, H.; Neutze, R.; Newstead, S.; Poolman, B.; Tate, C. G.; Vogel, H., Overcoming barriers to membrane protein structure determination. Nat. Biotechol. 2011, 29, 335-340. (5) Gao, Y.; Cao, E. H.; Julius, D.; Cheng, Y. F., TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 2016, 534, 347-351. (6) Hubbell, W. L.; Cafiso, D. S.; Altenbach, C., Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol. 2000, 7, 735-739. (7) Sezgin, E.; Schwille, P., Fluorescence techniques to study lipid dynamics. Cold Spring Harb. Perspect. Biol. 2011, 311, a009803. (8) Liang, B. Y.; Tamm, L. K., NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nat. Struct. Mol. Biol. 2016, 23, 468-474. (9) Opella, S. J., Solid-state NMR and membrane proteins. J Magn. Reson. 2015, 253, 129-137. (10) Tzakos, A. G.; Grace, C. R.; Lukavsky, P. J.; Riek, R., NMR techniques for very large proteins and rnas in solution. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 319-342. (11) Baker, K. A.; Tzitzilonis, C.; Kwiatkowski, W.; Choe, S.; Riek, R., Conformational dynamics of the KcsA potassium channel governs gating properties. Nat. Struct. Mol. Biol. 2007, 14, 1089-95. (12) Hiller, S.; Garces, R. G.; Malia, T. J.; Orekhov, V. Y.; Colombini, M.; Wagner, G., Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 2008, 321, 1206-1210. (13) Elter, S.; Raschle, T.; Arens, S.; Viegas, A.; Gelev, V.; Etzkorn, M.; Wagner, G., The use of amphipols for NMR structural characterization of 7-TM proteins. J. Membr. Biol. 2014, 247, 957-964. (14) Wylie, B. J.; Bhate, M. P.; McDermott, A. E., Transmembrane allosteric coupling of the gates in a potassium channel. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 185-190. (15) Wang, S.; Munro, R. A.; Shi, L.; Kawamura, I.; Okitsu, T.; Wada, A.; Kim, S. Y.; Jung, K. H.; Brown, L. S.; Ladizhansky, V., Solid-state NMR spectroscopy structure determination of a lipidembedded heptahelical membrane protein. Nat. Methods 2013, 10, 1007-1012.

As there is a growing number of membrane proteins which are embedded in lipid nanodiscs and studied by solution-state NMR or cryo-EM, our data indicate that those studies can be extended also towards protondetected solid-state NMR under fast MAS.

ASSOCIATED CONTENT Supporting Information. Details on sample preparation, NMR experimental parameters, data on detailed spectra analysis, 1H and 15N R’2 coherence decay rates, temperature-dependent series of TROSY 15 1 spectra, bulk N and H T1ρ data This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author a.Bö[email protected] [email protected] [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

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R72

10 5 0

-5

-5

[ppm] 10.5 9.5Plus Environment 8.5 7.5 ACS Paragon 1 δ( H)

[ppm] 9.8

9.4

9.0 1 δ( H)

8.6

8.2

[ppm]

Page 15 of 16 105 1 2 3 110 4 5 6 115 7 15 8 9 120 10 11 12 125 13 14 15 130 16 17 18 19 20 21 22 23 105 24 25 26 110 27 28 29 115 30 31 15 32 120 33 34 35 125 36 37 38 130 39 40 41 42 43 44

B

A

The Journal 20 of °CPhysical Chemistry 105 110 115

δ( N)

[ppm]

20 °C

120 125 130


protonated

solid

9.5

9.0

8.5

C

8.0

7.5

7.0

6.5

10.5 10.0

9.5

solid 9.0

8.5

8.0

7.5

D

20˚C

7.0

6.5

20˚C

105 110 115

δ( N)

120 125 130


solution 9.5

9.0

8.5

δ(1H)

protonated

Paragon 10.5 10.0 8.0ACS 7.5 7.0 Plus 6.5 Environment [ppm]

9.5

solution 9.0

8.5

δ(1H)

8.0

7.5

7.0 6.5 [ppm]


A

18 °C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 1 52 53 54 0.8 55 56 57 0.6 58 59 60

Page 16 of 16

[a.u.]

18 °C

32 °C

19.5 °C

33 °C

20.5 °C

34 °C

21 °C 22 °C

8

22 °C 23 °C 24 °C

24 °C

6

25 °C

I

26 °C 26.5 °C

4

27.5 °C 28 °C 29 °C 30 °C

32 °C

2

31.5 °C

0 10

9

8

7

δ(1H)

B

[ppm]

6

19.5 °C 26.5 °C

33 °C

39 °C

42.5 °C

[a.u.]

19.5 °C

33 °C

20 °C

34 °C

21 °C

35 °C

22 °C

35.5 °C

23 °C

36.5 °C

24 °C

37.5 °C

25 °C

38.5 °C

26 °C

39 °C

26.5 °C

40 °C

27.5 °C

42.5 °C

28.5 °C

43 °C

29.5 °C

44 °C

30 °C

45 °C

8

6

I 4

31 °C

2

32 °C

0 10

9

8

δ(1H)

C

7

[ppm]

6

D 1

1

1

0.8

0.8

0.8

0.6

0.6

0.6

0.4

0.4

0.2

0.2

IN

0.4

0.4

0.2

0.2

0

0 10

15

20

25

30

T

35

40

45 [°C]

0

0 10

15

20


25

30

T

35

40

45

[°C]

IN

Proton-Detected NMR Spectroscopy of Nanodisc-Embedded

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