Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy

Abstract

Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank. Solid-state Nuclear Magnetic Resonance (SSNMR) has long been considered as an attractive alternative because it allows for studies of membrane proteins in both native-like membranes composed of synthetic lipids and in cell membranes. Over the past decade, SSNMR has been rapidly developing into a major structural method, and a growing number of membrane protein structures obtained by this technique highlights its potential. Here we discuss membrane protein sample requirements, review recent progress in SSNMR methodologies, and describe recent advances in characterizing membrane proteins in the environment of a cellular membrane.

Keywords: cell membrane; lipid bilayer; membrane protein; protein structure; solid-state NMR.

Figure 1

Solid-state NMR approaches to membrane protein structure determination. (A) Protein molecules can be uniformly oriented with respect to the magnetic field B_o NMR spectra correlating the ¹H-¹⁵N dipolar coupling to the ¹⁵N chemical shift show a circular pattern, which reports on the topology of the protein andits backbone structure. An example on the left is a spectrum of the transmembrane domain of virus protein “u” comprising residues 1–32. Reprinted from Journal of Molecular Biology, 333, S.H. Park, A.A. Mrse, A.A. Nevzorov M. F. Mesleh, M. Oblatt-Montal, M. Montal and S.J. Opella, “Three-dimensional Structure of the Channel-forming Trans-membrane Domain of Virus Protein “u” (Vpu) from HIV-1” (2003) 409–424. Copyright 2003, with permission from Elsevier. (B) Magic angle spinning (MAS) NMR is applied to unoriented samples. MAS averages out anisotropic interactions to their isotropic values, and results in spectra that resemble those from samples in solution phase. An example on the right is a 2D NCA spectrum of Anabaena Sensory Rhodopsin correlating isotropic chemical shifts of ¹⁵N and ¹³C_α atoms. Reproduced with permission from Angewandte Chemie Int Ed., ‘‘Conformation of a seven-helical transmembrane photosensor n the lipid environment’’ 50 (2011) 1302–1305. Copyright 2011 John Wiley & Sons, Inc. Additionally, in the case when a protein undergoes fast (<10⁻⁶ s) rotational diffusion, correlating the ¹H-¹⁵N dipolar couplings to the ¹⁵N chemical shift would result in circular patterns similar to that shown on the left (Rotational Alignment), and can be used as an independent way to obtain backbone structure, or combined with distance measurements under MAS conditions.

Figure 2

Magic angle spinning carbon-carbon correlation spectra of various membrane proteins. (A) 900 MHz spectrum of trimeric YadA. Reprinted by permission from Macmillan Publishers Ltd: Scientific Reports, 2, S.A. Shahid, S. Markovic, D. Linke, B.J. van Rossum, “Assignment and secondary structure of the YadA membrane protein by solid-state MAS NMR”, (2012) 803. Copyright 2012. (B) 800 MHz spectrum of chimeric potassium channel KcsA-Kv1.3. Adapted with permission from J. Am. Chem. Soc., 130, R. Schneider, C. Ader, A. Lange, K. Giller, S. Hornig, O. Pongs, S. Becker, M. Baldus, “Solid-state NMR spectroscopy applied to a chimeric potassium channel in lipid bilayers” (2008) 7427–7435. Copyright 2008 American Chemical Society. (C) 750 MHz spectrum of ⁴⁰DsbB. Reprinted from Journal of Molecular Biology, 425, M. Tang, A.E. Nesbitt, L.J. Sperling, D.A. Berthold, C.D. Schwieters, R.B. Gennis, C.M. Rienstra, “Structure of the disulfide bond generating membrane protein DsbB in the lipid bilayer” (2013) 1670–1682. Copyright 2013, with permission from Elsevier. (D) 800 MHz spectrum of proteorhodopsin (unpublished data). (E) 900 MHz spectrum of VDAC1 sample prepared as 2D crystals.⁸⁶ Adapted with permission from J. Am. Chem. Soc., 134, M.T. Eddy, T.C. Ong, L. Clark, O. Tejido, P.C.van der Wel, R. Garces, G. Wagner, T.K. Rostovtseva, R.G. Griffin, “Lipid dynamics and protein-lipid interactions in 2D crystals formed with the ß-barrel integral membrane protein VDAC1” Copyright 2012 American Chemical Society. (F) 800 MHz spectrum of water channel human aquaporin 1.⁵³ Reprinted from Journal of Biomolecular NMR, S. Emami, Y. Fan, R. Munro, V. Ladizhansky, L.S. Brown, “Yeast-expressed human membrane protein aquaporin-1 yields excellent resolution of solid-state MAS NMR spectra” 55, (2013) 147155. Reproduced with kind permission from Springer Science and Business Media.

Figure 3

Sample preparation strategy for cellular NMR of PagL employed in Ref. 47. A: Schematic representation of the E. coli K-12 cell envelope. B: Preparation of whole cell, cell envelope, and proteoliposome samples. In the initial step, cells are first grown on the unlabeled medium, and then transferred to isotopically labeled medium for induction. Adapted with permission from Ref. 47, The National Academy of Sciences of the USA, 2012.

Figure 4

¹³C-¹³C correlation spectra of whole cell (A), cell envelope (B) and proteoliposome (C) samples containing PagL. Encircled regions demonstrate characteristic alanines, serines and threonines of PagL. Adapted with permission from Ref. (47), The National Academy of Sciences of the USA, 2012.

Figure 5

Sample preparation of ASR in E. coli membranes. Following growth in unlabeled media to the logarithmic phase (Step 1), cells are resuspended in isotopically enriched media at a high concentration, and protein expression is induced (Step 2). Cells are broken in Step 3, subjected to sucrose gradient to separate inner and outer membranes (Step 4) and to 2 phase purification system to isolate membrane fractions containing the His-tagged ASR (Step 5). Proteoliposomes are represented as green circles, ASR is shown as red insertions.

Figure 6

ASR in E. coli membrane. A: 16 peaks could be assigned from comparison of 2D NCA spectra of the proteoliposome and E. coli sample (the latter is shown in A). B−D: Much higher resolution was obtained using 3D CANCO and NCACB spectroscopy, which permitted assignments for ∼40% of the protein residues. Red and black contours in the representative 2D planes represent peaks detected in the proteoliposome and E. coli sample, respectively. Blue contours are from the control sample containing the background proteins, but not ASR. E,F: Structural conservation in ASR in E. coli vs. proteoliposomes. In E, residues surrounding retinal are well conserved and shown in green. F: Residues at the intermonomer interface are well conserved (shown in green). Small chemical shift perturbations (up to 1 ppm for ¹⁵N and up to 0.5 ppm for ¹³C) are detected in some of the residues in the helices forming oligomerization interface but facing interior of the protein or lipids (shown in yellow). Reprinted from Biophysical Journal, 108, 7, 1683–1696, M. Ward, S. Wang, R. Munro, E. Ritz, I. Hung, P. Gor'kov, Y. Jiang, H. Liang, L. Brown, V. Ladizhansky, “In situ Structural Studies of Anabaena Sensory Rhodopsin in the E. coli Membrane”, with permission from Elsevier.

Figure 7

Selected structures of membrane proteins determined or refined using solid-state NMR restraints. Protein name, molecular weight, lipid mimetic used, method and PDB code are given for each structure. Different colors are used to differentiate individual subunits.