Proton-Based Structural Analysis of a Heptahelical Transmembrane Protein in Lipid Bilayers

Abstract

The structures and properties of membrane proteins in lipid bilayers are expected to closely resemble those in native cell-membrane environments, although they have been difficult to elucidate. By performing solid-state NMR measurements at very fast (100 kHz) magic-angle spinning rates and at high (23.5 T) magnetic field, severe sensitivity and resolution challenges are overcome, enabling the atomic-level characterization of membrane proteins in lipid environments. This is demonstrated by extensive 1H-based resonance assignments of the fully protonated heptahelical membrane protein proteorhodopsin, and the efficient identification of numerous 1H–1H dipolar interactions, which provide distance constraints, inter-residue proximities, relative orientations of secondary structural elements, and protein–cofactor interactions in the hydrophobic transmembrane regions. These results establish a general approach for high-resolution structural studies of membrane proteins in lipid environments via solid-state NMR.

Structural Studies of Self‐Assembled Subviral Particles: Combining Cell‐Free Expression with 110 kHz MAS NMR Spectroscopy

“…Theg lobal T 2 ' time measured on as eries of 1D spectra was 3.9 AE 0.6 ms,a nd the spin-lattice relaxation time in the rotating frame T 11 = 18 AE 3ms ( Table S1). Thel inewidths of DHBs Sa re larger than the linewidths of microcrystalline proteins such as ubiquitin, [28] SH3, [29] or GB1, [30] but are found to be close to the ones observed for deuterated, HN back-exchanged, and fully protonated model membrane proteins,s uch as OmpG [29] and proteorhodopsin, [14] which show proton linewidths of around 100 and 190 Hz, respectively.W hile deuteration is always beneficial in terms of linewidth, it only reduces the coherent linewidth, and depending on the size of incoherent contributions,t he effect can be larger or smaller.T oseparate the effects as much as possible, we therefore used only perdeuterated proteins in this study. 1D traces along different nitrogen dimensions are shown in Figure 3toi llustrate the signals in the proton dimension and their signal-to-noise ratios.T he signals in the spectrum are distributed over as pectral width that can be expected for af ully a-helical protein, which intrinsically shows as maller chemical-shift dispersion than proteins containing b-strands or both a-helical and b-strand conformations.I ndeed, as [25] (c) or aDHBs S-specific antibody [26] (d).…”

“…Figure S6 shows the spectrum processed with other apodization functions,i no rder to illustrate the artificial resolution enhancement that can be achieved. Thel inewidths of DHBs Sa re larger than the linewidths of microcrystalline proteins such as ubiquitin, [28] SH3, [29] or GB1, [30] but are found to be close to the ones observed for deuterated, HN back-exchanged, and fully protonated model membrane proteins,s uch as OmpG [29] and proteorhodopsin, [14] which show proton linewidths of around 100 and 190 Hz, respectively.W hile deuteration is always beneficial in terms of linewidth, it only reduces the coherent linewidth, and depending on the size of incoherent contributions,t he effect can be larger or smaller.T oseparate the effects as much as possible, we therefore used only perdeuterated proteins in this study. Theg lobal T 2 ' time measured on as eries of 1D spectra was 3.9 AE 0.6 ms,a nd the spin-lattice relaxation time in the rotating frame T 11 = 18 AE 3ms ( Table S1).…”

“…[10] TheDHBV Sprotein is similar to that of HBV,b ut lacks the cysteine-rich antigenic loop of HBs S, reducing the total size of DHBs Sto167 amino acid residues versus 226 residues for HBs S( see Figure S2 for sequence alignments and Figure 1b for ap redicted model of S). While > 100 kHz MAS NMR spectroscopy, [11][12][13][14][15] cell-free expression, [16][17][18] as well as NMR analyses of large assemblies [19,20] and membrane proteins [21,22] have been demonstrated before,w eh ere show that despite the already high complexity hidden behind every single one of these elements,t heir combination is possible, and allowed us to obtain NMR data of DHBV self-assembled SVPs with suitable resolution and sensitivity.T his method should lead the way for investigations of these,a nd similar, large membrane protein assemblies with currently intractable structures. While > 100 kHz MAS NMR spectroscopy, [11][12][13][14][15] cell-free expression, [16][17][18] as well as NMR analyses of large assemblies [19,20] and membrane proteins [21,22] have been demonstrated before,w eh ere show that despite the already high complexity hidden behind every single one of these elements,t heir combination is possible, and allowed us to obtain NMR data of DHBV self-assembled SVPs with suitable resolution and sensitivity.T his method should lead the way for investigations of these,a nd similar, large membrane protein assemblies with currently intractable structures.…”

“…Whereas CF HBs Sexpression still needs further optimization as the yields are rather low and may require the presence of chaperones to keep the protein in solution (data not shown), the DHBV protein expressed well in the presence of am ild detergent and assembled spontaneously.T ogether with its reduced size,t his feature makes DHBV SVPs ah ighly attractive model system for structural studies by solid-state NMR spectroscopy.I mportantly,t he recent development of > 100 kHz magic angle spinning (MAS) solid-state NMR proton detection techniques that reduce the requested sample amount by nearly two orders of magnitude opens the possibility to analyze the protein amounts typically obtained in cell-free expression (see below). While > 100 kHz MAS NMR spectroscopy, [11][12][13][14][15] cell-free expression, [16][17][18] as well as NMR analyses of large assemblies [19,20] and membrane proteins [21,22] have been demonstrated before,w eh ere show that despite the already high complexity hidden behind every single one of these elements,t heir combination is possible, and allowed us to obtain NMR data of DHBV self-assembled SVPs with suitable resolution and sensitivity.T his method should lead the way for investigations of these,a nd similar, large membrane protein assemblies with currently intractable structures.…”

Proton‐Detected Solid‐State NMR of the Cell‐Free Synthesized α‐Helical Transmembrane Protein NS4B from Hepatitis C Virus

“…The increase in MAS frequency resulted in a concomitant decrease in sample amount, to below the milligram, which presents a reduction in protein of about a factor 100 compared to 13 C detection, that is roughly compensated by the sensitivity gain inherent to 1 H‐detection techniques . The first 60 to 100 kHz MAS schemes have been applied to well‐established membrane protein systems, such as proteorhodopsin, outer membrane beta‐barrels, VDAC, a truncated variant of influenza A M2 channel, KcsA or BamA . Sample preparation of these proteins mainly followed previously established protocols using 13 C detection, and included formation of 2D crystals or reconstitution into liposomes at low lipid‐to‐protein ratio (LPR, w / w ) by dialysis .…”