REDOR NMR on a hydrophobic peptide in oriented membranes

A method is presented for the calculation of REDOR dephasing for specifically labeled membrane-spanning peptides in uniformly aligned lipid bilayers under magic angle oriented sample spinning (MAOSS) conditions. Numerical simulations are performed for dephasing of (13)C signal by (15)N when the labels are placed in an alpha-helical peptide at the carbonyl of residue (i) and amide nitrogen of residue (i + 2) to show the dependency of REDOR echo intensity on the peptide tilt angle relative to the membrane normal. The approach was applied to the labeled transmembrane domain of phospholamban ([(15)N-Leu(37), (13)C-Leu(39)]PLBTM) incorporated into dimyristoylphosphatidylcholine bilayers. The dephasing observed for a random membrane dispersion showed that the peptide was alpha-helical in the region including the two labels, and dephasing in oriented membranes showed that the peptide helix was tilted by 25 degrees +/- 7 degrees relative to the bilayer normal. These results agree with those obtained by other spectroscopic methods.     

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

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.     

Determination of membrane Peptide orientation by 1H-detected 2H NMR spectroscopy

We demonstrate the application of the proton inverse detected deuteron (PRIDE) NMR technique to the measurement of the orientation of membrane-bound peptides with enhanced sensitivity. Gramicidin D, a transmembrane peptide, and ovispirin, a surface-bound peptide, were used as model systems. The peptides were 2H-labeled by 1H/2H exchange and oriented uniaxially on glass plates. The directly detected 2H spectra of both peptides showed only a strong D(2)O signal and no large quadrupolar splittings. In contrast, the PRIDE spectrum of gramicidin exhibited quadrupolar splittings as large as 281 kHz, consistent with its transmembrane orientation. Moreover, the large D(2)O signal in the directly detected 2H spectra was cleanly suppressed in the PRIDE spectrum. For ovispirin, the 1H indirectly detected 2H spectrum revealed a 104 kHz splitting and a zero-frequency peak. The former reflects the in-plane orientation of most of the helix axis, while the latter results from residues with a magic-angle orientation of the N-D bonds. These are consistent with previous 15N NMR results on ovispirin. The combination of PRIDE and exchange labeling provides an economical and sensitive method of studying membrane peptide orientations in lipid bilayers without the influence of D(2)O and with the ability to detect N-D bonds at the magic angle from the bilayer normal.     

'q-Titration' of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy

'q-Titration' refers to the systematic comparison of signal intensities in solution NMR spectra of uniformly (15)N labeled membrane proteins solubilized in micelles and isotropic bicelles as a function of the molar ratios (q) of the long-chain lipids (typically DMPC) to short-chain lipids (typically DHPC). In general, as q increases, the protein resonances broaden and correspondingly have reduced intensities due to the overall slowing of protein reorientation. Since the protein backbone signals do not broaden uniformly, the differences in line widths (and intensities) enable the narrower (more intense) signals associated with mobile residues to be differentiated from the broader (less intense) signals associated with "structured" residues. For membrane proteins with between one and seven trans-membrane helices in isotropic bicelles, we have been able to find a value of q between 0.1 and 1.0 where only signals from mobile residues are observed in the spectra. The signals from the structured residues are broadened so much that they cannot be observed under standard solution NMR conditions. This q value corresponds to the ratio of DMPC:DHPC where the signals from the structured residues are "titrated out" of the spectrum. This q value is unique for each protein. In magnetically aligned bilayers (q>2.5) no signals are observed in solution NMR spectra of membrane proteins because the polypeptides are "immobilized" by their interactions with the phospholipid bilayers on the relevant NMR timescale (～10(5)Hz). No signals are observed from proteins in liposomes (only long-chain lipids) either. We show that it is feasible to obtain complementary solution NMR and solid-state NMR spectra of the same membrane protein, where signals from the mobile residues are present in the solution NMR spectra, and signals from the structured residues are present in the solid-state NMR spectra. With assigned backbone amide resonances, these data are sufficient to describe major features of the secondary structure and basic topology of the protein. Even in the absence of assignments, this information can be used to help establish optimal experimental conditions.     

NMR-Based amide hydrogen-deuterium exchange measurements for complex membrane proteins: development and critical evaluation

A method for measuring site-specific amide hydrogen-deuterium exchange rates for membrane proteins in bilayers is reported and evaluated. This method represents an adaptation and extension of the approach of Dempsey and co-workers (Biophys. J. 70, 1777-1788 (1996)) and is based on reconstituting (15)N-labeled membrane proteins into phospholipid bilayers, followed by lyophilization and rehydration with D(2)O or H(2)O (control). Following incubation for a time t under hydrated conditions, samples are again lyophilized and then solubilized in an organic solvent system, where (1)H-(15)N HSQC spectra are recorded. Comparison of spectra from D(2)O-exposed samples to spectra from control samples yields the extent of the H-D exchange which occurred in the bilayers during time t. Measurements are site specific if specific (15)N labeling is used. The first part of this paper deals with the search for a suitable solvent system in which to solubilize complex membrane proteins in an amide "exchange-trapped" form for NMR quantitation of amide peak intensities. The second portion of the paper documents application of the overall procedure to measuring site-specific amide exchange rates in diacylglycerol kinase, a representative integral membrane protein. Both the potential usefulness and the significant limitations of the new method are documented.     

Rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state NMR spectroscopy

Solid-state NMR experiments on mechanically aligned bilayer and magnetically aligned bicelle samples demonstrate that membrane proteins undergo rapid rotational diffusion about the normal in phospholipid bilayers. Narrow single-line resonances are observed from 15N labeled sites in the trans-membrane helix of the channel-forming domain of the protein Vpu from HIV-1 in phospholipid bilayers with their normals at angles of 0 degrees, 20 degrees, 40 degrees, and 90 degrees, and bicelles with their normals at angles of 0 degrees and 90 degrees with respect to the direction of the applied magnetic field. This could only occur if the entire polypeptide undergoes rotational diffusion about the bilayer normal. Comparisons between experimental and simulated spectra are consistent with a rotational diffusion coefficient (DR) of approximately 10(5)s-1.     

Three-dimensional solid-state NMR spectroscopy is essential for resolution of resonances from in-plane residues in uniformly (15)N-labeled helical membrane proteins in oriented lipid bilayers

Uniformly (15)N-labeled samples of membrane proteins with helices aligned parallel to the membrane surface give two-dimensional PISEMA spectra that are highly overlapped due to limited dispersions of (1)H-(15)N dipolar coupling and (15)N chemical shift frequencies. However, resolution is greatly improved in three-dimensional (1)H chemical shift/(1)H-(15)N dipolar coupling/(15)N chemical shift correlation spectra. The 23-residue antibiotic peptide magainin and a 54-residue polypeptide corresponding to the cytoplasmic domain of the HIV-1 accessory protein Vpu are used as examples. Both polypeptides consist almost entirely of alpha-helices, with their axes aligned parallel to the membrane surface. The measurement of three orientationally dependent frequencies for Val17 of magainin enabled the three-dimensional orientation of this helical peptide to be determined in the lipid bilayer.     

Probing membrane topology by high-resolution 1H-13C heteronuclear dipolar solid-state NMR spectroscopy

Membrane topology changes introduced by the association of biologically pertinent molecules with membranes were analyzed utilizing the (1)H-(13)C heteronuclear dipolar solid-state NMR spectroscopy technique (SAMMY) on magnetically aligned phospholipid bilayers (bicelles). The phospholipids (1)H-(13)C dipolar coupling profiles lipid motions at the headgroup, glycerol backbone, and the acyl chain region. The transmembrane segment of phospholamban, the antimicrobial peptide (KIGAKI)(3) and cholesterol were incorporated into the bicelles, respectively. The lipids (1)H-(13)C dipolar coupling profiles exhibit different shifts in the dipolar coupling contour positions upon the addition of these molecules, demonstrating a variety of interaction mechanisms exist between the biological molecules and the membranes. The membrane topology changes revealed by the SAMMY pulse sequence provide a complete screening method for analyzing how these biologically active molecules interact with the membrane.     

Transmembrane helix tilting: insights from calculating the potential of mean force

To explore the microscopic forces governing the helix tilting in membranes, we have calculated the potential of mean force (PMF) as a function of tilt angle (tau) of WALP19, a transmembrane model peptide, in a dimyristoylphosphatidylcholine membrane. The PMF shows a wide range of thermally accessible tilt angles (5 degrees to 22 degrees ) with a minimum at tau=12.5 degrees . The free energy decomposition reveals that the helix tilting up to tau=12.5 degrees is mostly driven by the entropy contribution arising from the helix precession around the membrane normal, whereas the PMF increase after tau=12.5 degrees results from helical deformation due to the sequence-specific helix-lipid interactions.     

Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR

We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear (15)N-(13)C(α) and homonuclear (13)C-(13)C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped (13)C(α) resonances by strategically labeling the preceding residue. This method is highly complementary to (13)C(i-1)(')-(15)N(i)-(13)C(i)(α) and (13)C(i-1)(α)-(15)N(i-1)-(13)C(i)(') experiments to distinguish inter-residue spin systems at a minimal cost to signal-to-noise.     

1H assisted 13C/15N heteronuclear correlation spectroscopy in oriented sample solid-state NMR of single crystal and magnetically aligned samples

(1)H-irradiation under mismatched Hartmann-Hahn conditions provides an alternative mechanism for carrying out (15)N/(13)C transfers in triple-resonance heteronuclear correlation spectroscopy (HETCOR) on stationary samples of single crystals and aligned samples of biopolymers, which improve the efficiency especially when the direct (15)N-(13)C dipolar couplings are small. In many cases, the sensitivity is improved by taking advantage of the (13)C(α) labeled sites in peptides and proteins with (13)C detection. The similarities between experimental and simulated spectra demonstrate the validity of the recoupling mechanism and identify the potential for applying these experiments to virus particles or membrane proteins in phospholipid bilayers; however, further development is needed in order to derive quantitative distance and angular constraints from these measurements.     

PISEMA powder patterns and PISA wheels

Resonance patterns observed in 2D PISEMA (polarization inversion spin exchange at magic angle) spectra from a transmembrane alpha-helix have been demonstrated to yield structural details of the protein. This paper presents a mathematical discussion of the PISEMA powder spectrum as the image in the frequency plane of a quadratic function from the sphere of unit vectors. The simplicity of this function allows easy calculation of the powder spectrum. Based on this analysis of powder patterns, four degeneracies are discussed which arise in determining possible orientations associated with PISA spectra. This paper also gives parametric equations for PISA wheels, which are specific patterns observed in PISEMA spectra of oriented peptides. These wheels are useful both in assigning the resonances and in determining the orientation of the helix with respect to the magnetic field. The union of these PISA wheels gives the entire powder spectrum.     

The roles of homonuclear line narrowing and the1H amide chemical shift tensor in structure determination of proteins by solid-state NMR spectroscopy

The seminal contributions of Ulrich Haeberlen to homonuclear line narrowing and the determination of¹H chemical shift tensors are crucial for protein structure determination by solid-state nuclear magnetic resonance spectroscopy. The¹H chemical shift is particularly important in spectra obtained on oriented samples of membrane proteins as a mechanism for providing dispersion among resonances that are not resolved with the¹H-¹⁵N dipolar coupling and¹⁵N chemical shift frequencies. This is demonstrated with three-dimensional experiments on uniformly¹⁵N-labeled samples of Magainin antibiotic peptide and the protein Vpu from HIV-1 in oriented lipid bilayers. These experiments enable resonances in two-dimensional¹H-¹⁵N dipolar coupling/¹⁵N chemical shift planes separated by¹H chemical shift frequencies to be resolved and analyzed. These three-dimensional spectra are compared to one-dimensional spectra of full-length Vpu, the cytoplasmic domain of Vpu, and Magainin, as well as to two-dimensional spectra of fd coat protein and Colicin El polypeptide. The¹H amide chemical shift tensor provides valuable structural information, and this is demonstrated with its contributions to orientational restrictions to one of the in-plane helical residues of Magainin.     

Polarity dependence of EPR parameters for TOAC and MTSSL spin labels: correlation with DOXYL spin labels for membrane studies

TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) is a nitroxyl amino acid that can be incorporated in the backbone of peptides. DOXYL (4,4-dimethyl-oxazolidine-1-oxyl) is a nitroxyl ring that can be attached rigidly at specific C-atom positions in the acyl chains of phospholipids. Spin-labelled phosphatidylcholines of the DOXYL type have been used previously to establish the transmembrane polarity profile in biological lipid bilayers [D. Marsh, Polarity and permeation profiles in lipid membranes, Proc. Natl. Acad. Sci. USA 87 (2001) 7777-7782]. Here, we determine the polarity dependence of the isotropic (14)N-hyperfine couplings, a(o)(N), and g-values, g(o), in a wide range of protic and aprotic media, for a TOAC-containing dipeptide (Fmoc-TOAC-Aib-OMe) and for a DOXYL-containing fatty acid (12-DOXYL-stearic acid). The correlation between datasets for TOAC and DOXYL nitroxides in the various solvents is used to establish the polarity profile for isotropic hyperfine couplings of TOAC in a transmembrane peptide. This calibration can be used to determine the location of TOAC at selected residue positions in a transmembrane or surface-active peptide. A similar calibration procedure is also applied to a(o)(N) and g(o) for the pyrroline methanethiosulphonate nitroxide (MTSSL) that is used in site-directed spin-labelling studies of membrane proteins.     

Aggregation Behavior of pHLIP in Aqueous Solution at Low Concentrations: A Fluorescence Study

pH (low) insertion peptide (pHLIP) is a 36-residue peptide derived from the third transmembrane helix of the membrane protein bacteriorhodopsin. The hydrophobicity of this peptide makes it prone to aggregation even at low concentrations, but this has not been studied in detail. In this work, we characterized monomeric and aggregated forms of pHLIP in aqueous solution (pH 8) at low concentrations (~μM) using fluorescence-based approaches, complemented by circular dichroism (CD) spectroscopy. We show here that monomeric and aggregated pHLIP display differential red edge excitation shift (REES) and CD spectra. These spectroscopic features allowed us to show that pHLIP aggregates even at low concentrations. A detailed knowledge of the aggregation behavior of pHLIP under these conditions will be useful for monitoring and quantifying its interaction with membranes.     

High-resolution solid-state NMR of anisotropically mobile molecules under very low-power H decoupling and moderate magic-angle spinning

We show that for observing high-resolution heteronuclear NMR spectra of anisotropically mobile systems with order parameters less than 0.25, moderate magic-angle spinning (MAS) rates of 11 kHz combined with ¹H decoupling at 1–2 kHz are sufficient. Broadband decoupling at this low ¹H nutation frequency is achieved by composite pulse sequences such as WALTZ-16. We demonstrate this moderate MAS low-power decoupling technique on hydrated POPC lipid membranes, and show that 1 kHz ¹H decoupling yields spectra with the same resolution and sensitivity as spectra measured under 50 kHz ¹H decoupling when the same acquisition times (50 ms) are used, but the low-power decoupled spectra give higher resolution and sensitivity when longer acquisition times (>150 ms) are used, which are not possible with high-power decoupling. The limits of validity of this approach are explored for a range of spinning rates and molecular mobilities using more rigid membrane systems such as POPC/cholesterol mixed bilayers. Finally, we show ¹⁵N and ¹³C spectra of a uniaxially diffusing membrane peptide assembly, the influenza A M2 transmembrane domain, under 11 kHz MAS and 2 kHz ¹H decoupling. The peptide ¹⁵N and ¹³C intensities at low-power decoupling are 70–80% of the high-power decoupled intensities. Therefore, it is possible to study anisotropically mobile lipids and membrane peptides using liquid-state NMR equipment, relatively large rotors, and moderate MAS frequencies.     

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C-15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.     

Towards quantitative measurements in solid-state CPMAS NMR: A Lee-Goldburg frequency modulated cross-polarization scheme

A new scheme combining a Lee-Goldburg (LG) sequence with frequency modulation is proposed for cross-polarization (LG-FMCP) in solid-state magic-angle-spinning nuclear magnetic resonance. During the CP contact time, the (1)H magnetization is spin-locked along the magic angle by the LG sequence and the irradiation offset of the S spins (e.g., (15)N) is modulated sinusoidally with a constant RF amplitude. It is shown experimentally that the LG sequence significantly lengthens the proton spin-lattice relaxation time in the tilted rotating frame and that the frequency modulation shortens the cross-polarization time for non-protonated S spins. As a result of substantially increasing the difference in these relaxation rates, the non-protonated and protonated S spins can be more efficiently and more uniformly polarized with a relatively long CP contact time, making quantitative CP measurements possible. A sample of (15)N-delta 1-L-histidine lyophilized from a solution of pH 6.3 and a (15)N-delta 1-L-His labeled transmembrane helical peptide in hydrated lipid bilayers were used to illustrate the advantages of this scheme.     

Solid-state 17O NMR spectroscopy of a phospholemman transmembrane domain protein: implications for the limits of detecting dilute 17O sites in biomaterials

The 17O-'diluted' glycine-14 sites in a phospholemman (PLM) transmembrane domain protein are characterized by solid-state 17O NMR spectroscopy. The PLM transmembrane domain is an alpha-helical tetramer unit of four 28-residue peptides and is rigidly embedded in a bilayer where each alpha-helix has an average tilt of 7.3 degrees against the membrane normal. The PLM sample investigated here consists of a high lipid/peptide molar ratio (25:1) with one glycine residue in each helix enriched to <40% (17)O; thus, this is a very dilute 17O-sample and is the most dilute 17O-membrane protein to date to be characterized by solid-state 17O NMR spectroscopy. Based on the spectral analysis of 17O magic angle spinning (MAS) at 14.1 and 18.8T, the PLM transmembrane domain protein consists of multiple crystallographic gly14 sites, suggesting that the tetramer protein is an asymmetric unit with either C2- or C1-rotational symmetry along the bilayer normal.     

Uniaxial Diffusional Narrowing of NMR Lineshapes for Membrane Proteins Reconstituted in Magnetically Aligned Bicelles and Macrodiscs

Structure and dynamics of membrane proteins can be effectively studied by oriented-sample solid-state nuclear magnetic resonance (NMR) techniques when the lipid bilayers are macroscopically aligned with respect to the main magnetic field. Magnetic alignment of the protein-containing membrane bilayer results from the negative susceptibility anisotropy of the lipid hydrocarbon interior yielding perpendicular sample alignment. At this orientation, while the uniformity of alignment represents an essential prerequisite for obtaining high-quality NMR spectra, further line narrowing is obtained by uniaxial motional averaging of the azimuthal parts of the chemical shift anisotropies and dipolar couplings. The motional averaging is brought about by uniaxial rotational diffusion of the protein molecules about the normal to the membrane surface, which is perpendicular to the magnetic field. Uniaxial averaging is efficient when the motion about the axis of alignment becomes sufficiently fast (on the timescale of the dipolar couplings and chemical shift anisotropies). Line narrowing under uniaxial rotation can be theoretically modeled using the stochastic Liouville equation. In this mini-review, we illustrate the method of uniaxial averaging for the relatively small Pf1 coat protein which exhibits excellent resolution in magnetically aligned bicelles due to its fast uniaxial diffusion and even superior resolution in large (30 nm) nanodiscs (macrodiscs) stabilized by a belt peptide. Spectra of Pf1 coat protein in polymer-stabilized macrodiscs, an alternative and more robust alignment media, are presented. We also report on preliminary spectra of a much larger protein—uniformly ¹⁵N labeled M1-M4 domain for the human acetylcholine receptor. While some spectral resolution is apparent, significantly broader linewidths emphasize the need for creating fast rotating discoidal membrane mimetics.