Oligomeric Structure, Dynamics, and Orientation of Membrane Proteins from Solid-State NMR
2006; Elsevier BV; Volume: 14; Issue: 12 Linguagem: Inglês
10.1016/j.str.2006.10.002
ISSN1878-4186
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoSolid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both α-helical and β sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information. Solid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both α-helical and β sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information. Solid-state NMR (SSNMR) spectroscopy is rapidly maturing as a useful method for determining the atomic-resolution structure of insoluble proteins such as membrane proteins in their natural biological environments. In addition to the static three-dimensional structure, the dynamics, the orientation relative to the membrane, and the oligomeric assembly of these membrane proteins are also actively studied via SSNMR methods. Here, we review the latest progress in SSNMR studies of (1) the oligomeric structure of membrane proteins, i.e., how proteins self-assemble into functional units in the membrane; (2) both the global and internal side chain motion of membrane proteins; (3) the membrane protein orientation; and (4) the location and depth of insertion of membrane proteins. These emphases complement a number of recent reviews on protein structure determination by SSNMR (Baldus, 2006Baldus M. Solid-state NMR spectroscopy: molecular structure and organization at the atomic level.Angew. Chem. Int. Ed. Engl. 2006; 45: 1186-1188Crossref PubMed Scopus (29) Google Scholar, Bechinger et al., 2004Bechinger B. Aisenbrey C. Bertani P. The alignment, structure and dynamics of membrane-associated polypeptides by solid-state NMR spectroscopy.Biochim. Biophys. Acta. 2004; 1666: 190-204Crossref PubMed Scopus (62) Google Scholar, Bockmann, 2006Bockmann A. Structural and dynamic studies of proteins by high-resolution solid-state NMR.C. R. Chim. 2006; 9: 381-392Crossref Scopus (22) Google Scholar, Huster, 2005Huster D. Investigations of the structure and dynamics of membrane-associated peptides by magic angle spinning NMR.Prog. NMR Spectrosc. 2005; 46: 79-107Abstract Full Text Full Text PDF Scopus (58) Google Scholar, McDermott, 2004McDermott A.E. Structural and dynamic studies of proteins by solid-state NMR spectroscopy: rapid movement forward.Curr. Opin. Struct. Biol. 2004; 14: 554-561Crossref PubMed Scopus (129) Google Scholar, Opella and Marassi, 2004Opella S.J. Marassi F.M. Structure determination of membrane proteins by NMR spectroscopy.Chem. Rev. 2004; 104: 3587-3606Crossref PubMed Scopus (352) Google Scholar, Prosser et al., 2006Prosser R.S. Evanics F. Kitevski J.L. Al-Abdul-Wahid M.S. Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins.Biochemistry. 2006; 45: 8453-8465Crossref PubMed Scopus (188) Google Scholar). The principal method that has been used to determine membrane protein orientation is the 15N-based two-dimensional experiment, PISEMA, in which the N-H dipolar coupling and 15N chemical shift anisotropy (CSA) of a macroscopically aligned membrane protein are correlated. This method is most suitable for α-helical proteins, since the 15N interactions are approximately parallel to the helical axis. However, a number of alternative spin interactions have also been developed and applied to extract membrane protein orientation. These methods allow for the study of both α-helical and β sheet proteins, and they can determine membrane protein orientational change due to varying conditions such as changes in protein concentration. Two-dimensional N-H dipolar and 15N correlation spectra of uniformly and selectively 15N-labeled proteins continue to yield new or refined orientation information on a number of membrane peptides and proteins, including the transmembrane (TM) domain of the virus protein "u" (Vpu) from HIV-1 (Park et al., 2003Park S.H. Mrse A.A. Nevzorov A.A. Mesleh M.F. Oblatt-Montal M. Montal M. Opella S.J. Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1.J. Mol. Biol. 2003; 333: 409-424Crossref PubMed Scopus (206) Google Scholar), the membrane-bound state of the fd coat protein (Marassi and Opella, 2003Marassi F.M. Opella S.J. Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints.Protein Sci. 2003; 12: 403-411Crossref PubMed Scopus (162) Google Scholar), the M2 ion channel proteins of the influenza A virus (Wang et al., 2001Wang J. Kim S. Kovacs F. Cross T.A. Structure of the the transmembrane region of the M2 protein H+ channel.Protein Sci. 2001; 10: 2241-2250Crossref PubMed Scopus (220) Google Scholar) and of the acetylcholine receptor (Opella et al., 1999Opella S.J. Marassi F.M. Gesell J.J. Valente A.P. Kim Y. Oblatt-Montal M. Montal M. Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy.Nat. Struct. Biol. 1999; 6: 374-379Crossref PubMed Scopus (294) Google Scholar), bacteriorhodopsin (Kamihira et al., 2005Kamihira M. Vosegaard T. Mason A.J. Straus S.K. Nielsen N.C. Watts A. Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy.J. Struct. Biol. 2005; 149: 7-16Crossref PubMed Scopus (52) Google Scholar), and a GPCR protein, CXCR1 (Park et al., 2006bPark S.H. Prytulla S. De Angelis A.A. Brown J.M. Kiefer H. Opella S.J. High-resolution NMR spectroscopy of a GPCR in aligned bicelles.J. Am. Chem. Soc. 2006; 128: 7402-7403Crossref PubMed Scopus (96) Google Scholar). As an example of the information content from this type of experiment, the 50 residue fd coat protein in the membrane was found to contain a surface-bound helix (residues 8–18) connected by a short loop (residues 19–20) to a TM helix (residues 21–45). When the protein is assembled into bacteriophage particles, the surface helix and the TM helix merge into one nearly ideal, straight helix. Moreover, the TM helix of the membrane-bound protein has a distinct kink at residues 38–40, changing the helix tilt angle from 26° to 16° (Marassi and Opella, 2003Marassi F.M. Opella S.J. Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints.Protein Sci. 2003; 12: 403-411Crossref PubMed Scopus (162) Google Scholar). This same kink is also present in the phage-bound protein. The TM domain of the HIV-1 Vpu protein was used to refine PISEMA analysis as well as to extract biophysical principles. When the N-H dipolar couplings are plotted with the residue number, a periodic "dipolar wave" is obtained that exhibits changes in the helix orientation more clearly than the PISEMA spectra themselves (Mascioni and Veglia, 2003Mascioni A. Veglia G. Theoretical analysis of residual dipolar coupling patterns in regular secondary structures of proteins.J. Am. Chem. Soc. 2003; 125: 12520-12526Crossref PubMed Scopus (38) Google Scholar, Mesleh et al., 2003Mesleh M.F. Lee S. Veglia G. Thiriot D.S. Marassi F.M. Opella S.J. Dipolar waves map the structure and topology of helices in membrane proteins.J. Am. Chem. Soc. 2003; 125: 8928-8935Crossref PubMed Scopus (90) Google Scholar). With this analysis, it was found that Vpu has a kink in the middle of the TM domain, causing the tilt angle to change by ∼3° in the DOPC/DOPG membrane (Park et al., 2003Park S.H. Mrse A.A. Nevzorov A.A. Mesleh M.F. Oblatt-Montal M. Montal M. Opella S.J. Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1.J. Mol. Biol. 2003; 333: 409-424Crossref PubMed Scopus (206) Google Scholar). When incorporated into membranes of different thicknesses, Vpu changes its tilt angle to minimize the hydrophobic mismatch. The average helix tilt angles are 18°, 27°, 35°, and 51°, respectively, in lipid membranes with 18:1, 14:0, 12:0, and 10:0 acyl chains (Park and Opella, 2005Park S.H. Opella S.J. Tilt angle of a trans-membrane helix is determined by hydrophobic mismatch.J. Mol. Biol. 2005; 350: 310-318Crossref PubMed Scopus (136) Google Scholar). Interestingly, the kink disappears in the thinner membranes with 14-carbon to 10-carbon chains. The macroscopic alignment techniques include both mechanical alignment on glass plates, usually with the alignment axis parallel to the magnetic field (B0), and magnetic alignment with bicelles, with the alignment axis perpendicular or parallel to B0. In the case of perpendicular bicelles, although the alignment axis is not along B0, orientationally resolved spectra are still obtained since the bicelle-protein complex normally undergoes fast uniaxial rotation diffusion around the bicelle axis (Tian et al., 1998Tian F. Song Z. Cross T.A. Orientational constraints derived from hydrated powder samples by two-dimensional PISEMA.J. Magn. Reson. 1998; 135: 227-231Crossref PubMed Scopus (18) Google Scholar). The bicelle approach was recently shown to give better-resolved spectra than glass plate samples (Figure 1) (De Angelis et al., 2004De Angelis A.A. Nevzorov A.A. Park S.H. Howell S.C. Mrse A.A. Opella S.J. High-resolution NMR spectroscopy of membrane proteins in aligned bicelles.J. Am. Chem. Soc. 2004; 126: 15340-15341Crossref PubMed Scopus (93) Google Scholar). Interestingly, the structure of the bicelle is still under debate: discs, perforated sheets, and elongated cylindrical micelles have been proposed (Gaemers and Bax, 2001Gaemers S. Bax A. Morphology of three lyotropic liquid crystalline biological NMR media studied by translational diffusion anisotropy.J. Am. Chem. Soc. 2001; 123: 12343-12352Crossref PubMed Scopus (133) Google Scholar, Harroun et al., 2005Harroun T.A. Koslowsky M. Nieh M.P. de Lannoy C.F. Raghunathan V.A. Katsaras J. Comprehensive examination of mesophases formed by DMPC and DHPC mixtures.Langmuir. 2005; 21: 5356-5361Crossref PubMed Scopus (89) Google Scholar, van Dam et al., 2004van Dam L. Karlsson G. Edwards K. Direct observation and characterization of DMPC/DHPC aggregates under conditions relevant for biological solution NMR.Biochim. Biophys. Acta. 2004; 1664: 241-256Crossref PubMed Scopus (186) Google Scholar, van Dam et al., 2006van Dam L. Karlsson G. Edwards K. Morphology of magnetically aligning DMPC/DHPC aggregates-perforated sheets, not disks.Langmuir. 2006; 22: 3280-3285Crossref PubMed Scopus (64) Google Scholar). (A–C) The protein is in (A) a glass plate sample with the alignment axis parallel to B0, in (B) flipped bicelles, in which the alignment axis is parallel to B0, and in (C) unflipped bicelles, in which the alignment axis is perpendicular to B0. (D) Diagram of an unflipped bicelle containing a TM helix (reproduced from De Angelis et al., 2004De Angelis A.A. Nevzorov A.A. Park S.H. Howell S.C. Mrse A.A. Opella S.J. High-resolution NMR spectroscopy of membrane proteins in aligned bicelles.J. Am. Chem. Soc. 2004; 126: 15340-15341Crossref PubMed Scopus (93) Google Scholar with permission). Recently, an alternative substrate-supported alignment technique that involves nanoporous anodic aluminum oxide (AAO) disks was developed (Chekmenev et al., 2005Chekmenev E.Y. Hu J. Gor'kov P.L. Brey W.W. Cross T.A. Ruuge A. Smirnov A.I. 15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.J. Magn. Reson. 2005; 173: 322-327Crossref PubMed Scopus (30) Google Scholar, Lorigan et al., 2004Lorigan G.A. Dave P.C. Tiburu E.K. Damodaran K. Abu-Baker S. Karp E.S. Gibbons W.J. Minto R.E. Solid-state NMR spectroscopic studies of an integral membrane protein inserted into aligned phospholipid bilayer nanotube arrays.J. Am. Chem. Soc. 2004; 126: 9504-9505Crossref PubMed Scopus (34) Google Scholar). When the AAO surface is perpendicular to B0, the lipid bilayer normal inside the nanotubes is also perpendicular to B0. These AAO membranes have better thermal conductivity, and they allow the external environment, such as pH and ion concentration, to be readily adjusted in membrane protein structural studies. Ulrich and coworkers have developed a novel 19F NMR strategy to determine membrane protein orientations. The approach uses the F-F dipolar coupling in CF3-Phg to determine membrane peptide orientation. The magnitude of the coupling is obtained from the splitting, while the sign of the dipolar coupling is obtained from the anisotropic chemical shift (Glaser et al., 2004Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Ulrich A.S. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels.J. Magn. Reson. 2004; 168: 153-163Crossref PubMed Scopus (92) Google Scholar). The advantages of 19F as an orientational probe include its large CSA and strong 19F-19F dipolar coupling, which give high angular resolution, its high sensitivity due to the large gyromagnetic ratio, and the lack of background signals in membrane proteins. The 19F spin needs to be incorporated into the protein chemically. The fluorinated residues that have been demonstrated so far include 4-19F-phenylglycine (Phg) (Afonin et al., 2003Afonin S. Glaser R.W. Berditchevskaia M. Wadhwani P. Guhrs K.H. Mollmann U. Perner A. Ulrich A.S. 4-fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides.ChemBioChem. 2003; 4: 1151-1163Crossref PubMed Scopus (112) Google Scholar), CF3-Phg (Glaser et al., 2004Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Ulrich A.S. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels.J. Magn. Reson. 2004; 168: 153-163Crossref PubMed Scopus (92) Google Scholar), CF3-Ala (Grage and Ulrich, 2000Grage S.L. Ulrich A.S. Orientation-dependent (19)F dipolar couplings within a trifluoromethyl group are revealed by static multipulse NMR in the solid state.J. Magn. Reson. 2000; 146: 81-88Crossref PubMed Scopus (36) Google Scholar), and 4-19F-Phe (Buffy et al., 2005Buffy J.J. Waring A.J. Hong M. Determination of peptide oligomerization in lipid membranes with magic-angle spinning spin diffusion NMR.J. Am. Chem. Soc. 2005; 127: 4477-4483Crossref PubMed Scopus (78) Google Scholar). Using glass-plate-aligned samples and 19F CSA and F-F dipolar coupling measurements, Ulrich and coworkers investigated the orientation and orientational changes of the α-helical antimicrobial peptide PGLa (Glaser et al., 2004Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Ulrich A.S. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels.J. Magn. Reson. 2004; 168: 153-163Crossref PubMed Scopus (92) Google Scholar, Glaser et al., 2005Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Afonin S. Strandberg E. Ulrich A.S. Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR.Biophys. J. 2005; 88: 3392-3397Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), the cyclic β sheet antimicrobial peptide gramicidin S (Salgado et al., 2001Salgado J. Grage S.L. Kondejewski L.H. Hodges R.S. McElhaney R.N. Ulrich A.S. Membrane-bound structure and alignment of the antimicrobial β-sheet peptide gramicidin S derived from angular and distance constraints by solid-state 19F-NMR.J. Biomol. NMR. 2001; 21: 191-208Crossref PubMed Scopus (111) Google Scholar), and a fusogenic peptide, B18 (Afonin et al., 2004Afonin S. Durr U.H. Glaser R.W. Ulrich A.S. 'Boomerang'-like insertion of a fusogenic peptide in a lipid membrane revealed by solid-state 19F NMR.Magn. Reson. Chem. 2004; 42: 195-203Crossref PubMed Scopus (58) Google Scholar). The magainin-analog PGLa exhibits an in-plane orientation at low P/L molar ratios (1:200), but this changes to an oblique orientation, with the helical axis at an angle of 123° with the bilayer normal, when the P/L ratio increases to 1:50 and higher (Figure 2). At intermediate concentrations, the peptide undergoes fast exchange between these two orientations (Glaser et al., 2004Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Ulrich A.S. Orientation of the antimicrobial peptide PGLa in lipid membranes determined from 19F-NMR dipolar couplings of 4-CF3-phenylglycine labels.J. Magn. Reson. 2004; 168: 153-163Crossref PubMed Scopus (92) Google Scholar, Glaser et al., 2005Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Afonin S. Strandberg E. Ulrich A.S. Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR.Biophys. J. 2005; 88: 3392-3397Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The tilt angle does not have the typical double degeneracy between τ and 180 − τ, because the 19F chemical shift spectra are modulated by the F-F dipolar coupling within the CF3 group of the CF3-Phg labels, thus yielding the sign of the dipolar coupling. The rotation angle of both orientations is consistent with the amphipathic character of the peptide, putting the hydrophilic side chains toward the membrane surface. In applying this 19F NMR technique, it is important to check that the biological function of the peptide is not adversely affected by the replacement of the natural residues with CF3-Phg or 4F-Phg. The high sensitivity of 19F NMR spectra allows for the study of membrane peptide orientation over a wide P/L range, thus directly testing the hypothesis of orientational change. As P/L increases from the left to the right, the helix orientation changes from in-plane (τ = 89°) to an obliquely tilted and inserted state (τ = 123°). The inserted state was hypothesized to be dimerized (reproduced from Glaser et al., 2005Glaser R.W. Sachse C. Durr U.H. Wadhwani P. Afonin S. Strandberg E. Ulrich A.S. Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR.Biophys. J. 2005; 88: 3392-3397Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar with permission). Methyl-deuterated Ala is a simple and robust orientational probe. The three-site jump of the Ala methyl group occurs around the Cα-Cβ axis; thus, the quadrupolar splitting reflects the orientation of the Cα-Cβ bond relative to the alignment axis without other complicating torsional motion of the side chain. Combined with 15N chemical shift constraints, the 2H probe has been used to determine the orientation of designed amphipathic peptides KL14 and KL26 and a hydrophobic peptide, hΦ19W (Aisenbrey and Bechinger, 2004bAisenbrey C. Bechinger B. Tilt and rotational pitch angle of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy.Biochemistry. 2004; 43: 10502-10512Crossref PubMed Scopus (60) Google Scholar). The KL peptides were found to be parallel to the membrane plane with a rotation angle that places the polar residues toward water and the hydrophobic residues toward the membrane, while hΦ19W was found to be a TM peptide. Since 2H NMR has comparable or higher sensitivity than 15N NMR, and since the acquisition of quadrupolar spectra can be much faster than 15N spectra due to the absence of 1H decoupling, this Ala-2H NMR is an appealing approach for orientation determination. However, a single-site 2H coupling is usually insufficient to obtain a unique solution to both the tilt and rotation angles of the peptide, similar to the degeneracy that would be present in 1D 15N spectra of singly labeled peptides (hence the need for multiple 15N labeling and two-dimensional PISEMA spectra). Thus, systematic site-specific deuteration and Ala mutation have recently been reported to restrain the global orientation of membrane peptides (Strandberg et al., 2006Strandberg, E., Tremouilhac, P., Wadhwani, P., and Ulrich, A.S. (2006). Realignment of membrane-bound antimicrobial peptides studied by solid-state 2H- and 19F-NMR. In Rocky Mountain Conference on Analytical Chemistry: Breckenridge, CO.Google Scholar). The 13CO chemical shift tensor is a useful probe of β sheet peptide orientation, because the σxx principal axis is aligned along the strand axis, while the σzz axis is perpendicular to the β sheet plane. Therefore, for a uniaxially aligned peptide with the bilayer normal parallel to the magnetic field, the observation of a frequency close to 245 ppm (σxx) indicates that the strand axis is parallel to the bilayer normal (a TM peptide, for example), while a peak close to 95 ppm (σzz) indicates that the β sheet plane normal is parallel to the bilayer normal (for example, an in-plane peptide). 13CO chemical shift constraints have been used to determine the orientation of PG-1 (Yamaguchi et al., 2002Yamaguchi S. Waring A. Hong T. Lehrer R. Hong M. Solid-State NMR investigations of peptide-lipid interaction and orientation of a β-sheet antimicrobial peptide, protegrin.Biochemistry. 2002; 41: 9852-9862Crossref PubMed Scopus (138) Google Scholar) and TP-I (Hong and Doherty, 2006Hong M. Doherty T. Orientation determination of membrane-disruptive proteins using powder samples and rotational diffusion: a simple solid-state NMR approach.Chem. Phys. Lett. 2006; 432: 296-300Crossref PubMed Scopus (38) Google Scholar), two disulfide-stabilized β sheet antimicrobial peptides. PG-1 was found to exhibit an oblique orientation, with the strand axis tilted by ∼55° from the normal of the DLPC membrane. TP-I, on the other hand, is much more parallel to the membrane plane, with the strand axis tilted by only ∼20° from the plane. This difference is consistent with the different insertion depths of the two peptides: PG-1 is well inserted into the hydrophobic center of the membrane (Buffy et al., 2003aBuffy J.J. Hong T. Yamaguchi S. Waring A. Lehrer R.I. Hong M. Solid-state NMR investigation of the depth of insertion of protegin-1 in lipid bilayers using paramagnetic Mn2+.Biophys. J. 2003; 85: 2363-2373Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), while TP-I lies at the glycerol backbone region, in contact with the top of the acyl chains, but not the membrane center (Doherty et al., 2006Doherty T. Waring A.J. Hong M. Membrane-bound conformation and topology of the antimicrobial peptide tachyplesin-I by solid-state NMR.Biochemistry. 2006; PubMed Google Scholar). These differences are attributed to the different amphipathic structures of the two peptides. PG-1 has a hydrophilic-hydrophobic separation along the strand axis; thus, the hydrophobic domain can insert in a TM fashion. TP-I, on the other hand, exhibits side chain amphipathicity below and above the β hairpin plane, but no backbone amphiphilicity, thus favoring an in-plane orientation. Rigid-body uniaxial rotational diffusion of membrane peptides and proteins has recently become a topic of interest due to its relevance for orientation determination and for understanding the complete physical state of the protein. As a result of the fluidity of the liquid-crystalline lipid bilayer, membrane proteins undergo Brownian motions in the two-dimensional membrane at rates given by the Saffman-Delbrück equation (Saffman and Delbruck, 1975Saffman P.G. Delbruck M. Brownian motion in biological membranes.Proc. Natl. Acad. Sci. USA. 1975; 72: 3111-3113Crossref PubMed Scopus (1281) Google Scholar):Dr=kT4πηr2h,(1) where η is the viscosity of the membrane, T is the absolute temperature, and r and h are the radius and height, respectively, of the diffusing cylinder in the membrane. This equation predicts that TM proteins with radii up to ∼12 Å should undergo uniaxial rotations that are fast compared to the 2H quadrupolar couplings of ∼125 kHz and thus should average the 2H NMR spectra. If 15N and 13C chemical shift interactions at magnetic field strengths of 9.4–14.1 Tesla are used to monitor motion, then these smaller interactions (up to ∼6 kHz) allow proteins with radii up to ∼40 Å to still remain in the fast motional limit. Thus, motionally averaged 15N and 13C chemical shift spectra are expected even for relatively large membrane proteins. Many experimental reports of such uniaxial rotational diffusion have been given. These include ion channel peptides such as gramicidin A (Ketchem et al., 1993Ketchem R.R. Hu W. Cross T.A. High resolution conformation of Gramicidine A in a lipid bilayer by solid-state NMR.Science. 1993; 261: 1457-1460Crossref PubMed Scopus (600) Google Scholar), designed synthetic peptides such as KL14 and hΦ19W (Aisenbrey and Bechinger, 2004aAisenbrey C. Bechinger B. Investigations of polypeptide rotational diffusion in aligned membranes by 2H and 15N solid-state NMR spectroscopy.J. Am. Chem. Soc. 2004; 126: 16676-16683Crossref PubMed Scopus (69) Google Scholar), antimicrobial peptides such as PG-1 and ovispirin (Yamaguchi et al., 2001Yamaguchi S. Huster D. Waring A. Lehrer R.I. Tack B.F. Kearney W. Hong M. Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state NMR.Biophys. J. 2001; 81: 2203-2214Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, Yamaguchi et al., 2002Yamaguchi S. Waring A. Hong T. Lehrer R. Hong M. Solid-State NMR investigations of peptide-lipid interaction and orientation of a β-sheet antimicrobial peptide, protegrin.Biochemistry. 2002; 41: 9852-9862Crossref PubMed Scopus (138) Google Scholar), and helical bundles such as the TM domain of the M2 protein of influenza A virus (Song et al., 2000Song Z. Kovacs F.A. Wang J. Denny J.K. Shekar S.C. Quine J.R. Cross T.A. Transmembrane domain of M2 protein from influenza A virus studied by solid-state 15N polarization inversion spin exchange at magic angle NMR.Biophys. J. 2000; 79: 767-775Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). These peptides can have both α-helical and β sheet conformation, and they can be either TM or parallel to the membrane plane. Therefore, rotational diffusion appears to be universal given a suitable size and viscosity of the membrane. In all cases reported, the uniaxial rotational diffusion is around the bilayer normal, which is the unique direction in the two-dimensional membrane. Using 2H quadrupolar couplings, C-H and N-H dipolar interactions, 13C and 15N chemical shift anisotropies, and 1H relaxation time measurements, Hong and coworkers characterized the rates and amplitude of the uniaxial rotational diffusion of the M2 TM peptide (M2TMP) of influenza A virus (Cady and Hong, 2006Cady, S.D., and Hong, M. (2006). Determining the orientation of rotationally diffusing membrane proteins using powder samples and magic-angle spinning: a 2H, 13C, and 15N solid-state NMR investigation of a transmembrane helical bundle. In Rocky Mountain Conference on Analytical Chemistry: Breckenridge, CO.Google Scholar). They found that the rotational diffusion depends on the sample preparation condition: mixing of peptide and lipid in organic solvents prior to the self-assembly of lipid bilayers promotes rotational diffusion, while direct mixing of the peptide with the lipid vesicle solution produces proteins without fast global rotation on the 2H NMR timescale. These suggest preferential aggregation of the peptide when bound to lipid vesicle solutions. As expected from Equation 1, membrane protein orientation affects the rotational diffusion time constants due to the quadratic dependence on r and the linear dependence on h. A surface-bound membrane peptide has a much larger radius than the same-sized protein in a TM orientation and thus should be less mobile. Indeed, KL26, a 26 residue amphipathic, in-plane helical peptide consisting of Lys and Leu residues, was shown by 15N chemical shift spectra and 2H NMR spectra to be immobilized on these timescales (Aisenbrey and Bechinger, 2004bAisenbrey C. Bechinger B. Tilt and rotational pitch angle of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy.Biochemistry. 2004; 43: 10502-10512Crossref PubMed Scopus (60) Google Scholar). The radius of the peptide is ∼39 Å. In comparison, the 25 residue M2TMP with a similar length rotates even in the tetrameric state because of its smaller r for diffusion (Song et al., 2000Song Z. Kovacs F.A. Wang J. Denny J.K. Shekar S.C. Quine J.R. Cross T.A. Transmembrane domain of M2 protein from influenza A virus studied by solid-state 15N polarization inversion spin exchange at magic angle NMR.Biophys. J. 2000; 79: 767-775Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The same KL peptide with only 14 residues (KL14), which also has an in-plane orientation, does undergo fast uniaxial rotational diffusion on the 15N CSA and 2H timescales. Thus, the upper size limit of surface-bound peptides for rotational diffusion lies between 21 Å and 39 Å. When membrane proteins are bound to lipid bicelles in solution (Prosser et al., 1998Prosser R.S. Hwang J.S. Vold R.R. Magnetically aligned phospholipid bilayers with positive ordering: a new model membrane system.Biophys. J. 1998; 74: 2405-2418Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, Sanders et al., 1994Sanders C.R. Hare B.J. Howard K.P. Prestegard J.H. Magnetically oriented phospholipid micelles as a tool for the study of membrane associated molecules.Prog. NMR Spectrosc. 1994; 26: 421-444Abstract Full Text PDF Scopus (379) Google Scholar), uniaxial rotational diffusion around the bicelle normal occurs as a result of th
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