Polarization Inversion Spin Exchange At The Magic Angle Research Articles

Investigating the Biophysical Basis of Mosaic Spread in Aligned Samples

Oriented solid-state NMR (O-SSNMR) is a powerful technique to probe the structure and dynamics of membrane proteins. One of the most popular experiments on aligned samples is PISEMA (polarization inversion spin exchange at the magic angle), which is most often used to correlate the 1H-15N dipolar coupling with the 15N anisotropic chemical shift in membrane proteins. The spectral patterns in PISEMA are directly sensitive to both the secondary structure geometry with respect to the lipid bilayer normal and the mosaic spread within aligned samples. The latter will result in an increased linewidth of the peaks. Using the drug transporter EmrE as a model protein we investigated conditions that can reduce the mosaic spread in aligned samples and therefore increase the spectral resolution. EmrE is an integral membrane protein with four transmembrane domains and 110 residues that forms an anti-parallel and asymmetric homodimer. Interestingly, we discovered that ether linked lipids as well as the presence of conservative mutations in EmrE can profoundly increase the spectral resolution and therefore sensitivity of the PISEMA experiment, which implies a direct reduction in the mosaic spread of the samples for O-SSNMR. These results improve the feasibility of employing aligned methodology to determine the tilt angles and probe the conformational dynamics of membrane proteins with respect to the lipid bilayer.

Simultaneous acquisition of 2D and 3D solid-state NMR experiments for sequential assignment of oriented membrane protein samples.

We present a new method called DAISY (Dual Acquisition orIented ssNMR spectroScopY) for the simultaneous acquisition of 2D and 3D oriented solid-state NMR experiments for membrane proteins reconstituted in mechanically or magnetically aligned lipid bilayers. DAISY utilizes dual acquisition of sine and cosine dipolar or chemical shift coherences and long living (15)N longitudinal polarization to obtain two multi-dimensional spectra, simultaneously. In these new experiments, the first acquisition gives the polarization inversion spin exchange at the magic angle (PISEMA) or heteronuclear correlation (HETCOR) spectra, the second acquisition gives PISEMA-mixing or HETCOR-mixing spectra, where the mixing element enables inter-residue correlations through (15)N-(15)N homonuclear polarization transfer. The analysis of the two 2D spectra (first and second acquisitions) enables one to distinguish (15)N-(15)N inter-residue correlations for sequential assignment of membrane proteins. DAISY can be implemented in 3D experiments that include the polarization inversion spin exchange at magic angle via I spin coherence (PISEMAI) sequence, as we show for the simultaneous acquisition of 3D PISEMAI-HETCOR and 3D PISEMAI-HETCOR-mixing experiments.

Determination of the orientational order parameter of the homologous series of 4-cyanophenyl 4-alkylbenzoate (n.CN) by different methods

The orientational order parameters of a homologous series of 4-cyanophenyl 4-alkylbenzoates have been determined at different temperatures from 13C-NMR, x-ray diffraction, optical birefringence, high resolution density and diamagnetic susceptibility anisotropy measurements. To determine the temperature dependence of the orientational order parameter from the 13C chemical shift values, the two-dimensional polarization inversion spin exchange at the magic angle (PISEMA) method was also used for the measurement of 13C–1H dipolar couplings at different sites in the oriented phase. The nematic order parameters determined from each of these methods have been compared. Apart from a slight shift in their values their trends with temperature are very similar. The differences among the results obtained by these five different methods have been discussed. From the high resolution density data, the values of the critical exponents near the TN−I transition are found to lie between the Ising model and tricritical behaviour.

Sensitivity Enhancement in Solid-State Separated Local Field NMR Experiments by the Use of Adiabatic Cross-Polarization

Measurement of dipolar couplings using separated local field (SLF) NMR experiment is a powerful tool for structural and dynamics studies of oriented molecules such as liquid crystals and membrane proteins in aligned lipid bilayers. Enhancing the sensitivity of such SLF techniques is of significant importance in present-day solid-state NMR methodology. The present study considers the use of adiabatic cross-polarization for this purpose, which is applied for the first time to one of the well-known SLF techniques, namely, polarization inversion spin exchange at the magic angle (PISEMA). The experiments have been carried out on a single crystal of a model peptide, and a dramatic enhancement in signal-to-noise up to 90% has been demonstrated.

Improved resolution in dipolar NMR spectra using constant time evolution PISEMA experiment

The atomic structure of small molecules and polypeptides can be attained from anisotropic NMR parameters such as dipolar couplings (DC) and chemical shifts (CS). Separated local field experiments resolve DC and CS correlations into two dimensions. However, crowded NMR spectra represent a significant obstacle for the complete resolution of these anisotropic parameters. Using the polarization inversion spin exchange at the magic angle (PISEMA) experiment as a foundation, we designed new pulse schemes that use a constant time evolution in the dipolar (indirect) dimension to measure DC and CS correlations at high resolution. We demonstrated this approach on a 4-pentyl-4′-cyanobiphenyl (5CB) liquid crystal sample, achieving a resolution enhancement ranging from 30% to 60% for the resonances in the dipolar dimension. These new experiments open the possibility of obtaining significant resolution enhancement for multidimensional NMR experiments carried out on oriented liquid crystalline samples as well as oriented membrane proteins.

1H– 13C hetero-nuclear dipole–dipole couplings of methyl groups in stationary and magic angle spinning solid-state NMR experiments of peptides and proteins

13C NMR of isotopically labeled methyl groups has the potential to combine spectroscopic simplicity with ease of labeling for protein NMR studies. However, in most high resolution separated local field experiments, such as polarization inversion spin exchange at the magic angle (PISEMA), that are used to measure 1H– 13C hetero-nuclear dipolar couplings, the four-spin system of the methyl group presents complications. In this study, the properties of the 1H– 13C hetero-nuclear dipolar interactions of 13C-labeled methyl groups are revealed through solid-state NMR experiments on a range of samples, including single crystals, stationary powders, and magic angle spinning of powders, of 13C 3 labeled alanine alone and incorporated into a protein. The spectral simplifications resulting from proton detected local field (PDLF) experiments are shown to enhance resolution and simplify the interpretation of results on single crystals, magnetically aligned samples, and powders. The complementarity of stationary sample and magic angle spinning (MAS) measurements of dipolar couplings is demonstrated by applying polarization inversion spin exchange at the magic angle and magic angle spinning (PISEMAMAS) to unoriented samples.

Influence of Whole-Body Dynamics on 15N PISEMA NMR Spectra of Membrane Proteins: A Theoretical Analysis

Membrane proteins and peptides exhibit a preferred orientation in the lipid bilayer while fluctuating in an anisotropic manner. Both the orientation and the dynamics have direct functional implications, but motions are usually not accessible, and structural descriptions are generally static. Using simulated data, we analyze systematically the impact of whole-body motions on the peptide orientations calculated from two-dimensional polarization inversion spin exchange at the magic angle (PISEMA) NMR. Fluctuations are found to have a significant effect on the observed spectra. Nevertheless, wheel-like patterns are still preserved, and it is possible to determine the average peptide tilt and azimuthal rotation angles using simple static models for the spectral fitting. For helical peptides undergoing large-amplitude fluctuations, as in the case of transmembrane monomers, improved fits can be achieved using an explicit dynamics model that includes Gaussian distributions of the orientational parameters. This method allows extracting the amplitudes of fluctuations of the tilt and azimuthal rotation angles. The analysis is further demonstrated by generating first a virtual PISEMA spectrum from a molecular dynamics trajectory of the model peptide, WLP23, in a lipid membrane. That way, the dynamics of the system from which the input spectrum originates is completely known at atomic detail and can thus be directly compared with the dynamic output obtained from the fit. We find that fitting our dynamics model to the polar index slant angles wheel gives an accurate description of the amplitude of underlying motions, together with the average peptide orientation.

Orientation of Single-Span Transmembrane Peptides Investigated by Independent Solid-State NMR Methods: GALA and PISEMA

Model peptides of the “WALP” family have been shown by deuterium solid-state NMR to adopt small average tilt angles in model membranes; however, molecular dynamics simulations have consistently predicted larger tilt angles. It has been argued that peptide molecular motion could potentially compromise the observed tilt angles deduced from solid-state NMR. It was therefore of interest to employ an independent technique to address the discrepancy between experimental and theoretical methods.Here we report the analysis of the average orientation of single-span transmembrane peptides acetyl-GXALW(LA)6LWLAXA-[ethanol]amide (XWALP23, where X = K or G) in mechanically aligned lipid bilayers using two distinct solid-state NMR methods. GALA (Geometric Analysis of Labeled Alanines) employs the 2H signals of labeled alanine side chains, while the PISEMA (Polarization Inversion Spin Exchange at the Magic Angle) technique is based upon the 15N-1H signals from the peptide backbone. Due to the different angles between the helical axis and the Cα-Cβ or N-H bond vectors, these methods are expected to provide different sensitivities toward the molecular motion.GALA analysis of XWALP23 orientation in DLPC, DMPC and DOPC revealed that both peptides are tilted with respect to the membrane normal, with the magnitude of tilt dependent on membrane thickness (8-17° for KWALP23 and 6-13° for GWALP23). For comparison, PISEMA experiments were performed in DLPC, where the tilt is highest. The results from the two independent NMR methods are similar, with the tilt difference not exceeding 3 degrees (Vostrikov, V. et al. 2008. J Am Chem Soc, 38:12584). Although molecular dynamics simulations have not yet been performed for XWALP23 peptides, such calculations would be of interest for comparison with the experimental results from 2H NMR and 15N NMR, and with existing simulations for WALP19 and WALP23.

Tailoring 13C labeling for triple-resonance solid-state NMR experiments on aligned samples of proteins.

In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different (13)C labeling schemes with the purpose of overcoming the interfering effects of (13)C-(13)C dipole-dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% (13)C in all sites and metabolically labeled from bacterial growth on media containing [2-(13)C]-glycerol or [1,3-(13)C]-glycerol enables direct (13)C detection in solid-state NMR experiments without the need for homonuclear (13)C-(13)C dipole-dipole decoupling. The (13)C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent (15)N-detected spectra. The isotopic labeling pattern was analyzed for [2-(13)C]-glycerol and [1,3-(13)C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored (13)C-labeled samples, in contrast to their failure with conventional 100% uniformly (13)C-labeled samples.

Structural Dynamics and Topology of Phospholamban in Oriented Lipid Bilayers Using Multidimensional Solid-State NMR

Phospholamban (PLN), a single-pass membrane protein, regulates heart muscle contraction and relaxation by reversible inhibition of the sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Studies in detergent micelles and oriented lipid bilayers have shown that in its monomeric form PLN adopts a dynamic L shape (bent or T state) that is in conformational equilibrium with a more dynamic R state. In this paper, we use solid-state NMR on both uniformly and selectively labeled PLN to refine our initial studies, describing the topology and dynamics of PLN in oriented lipid bilayers. Two-dimensional PISEMA (polarization inversion spin exchange at the magic angle) experiments carried out in DOPC/DOPE mixed lipid bilayers reveal a tilt angle of the transmembrane domain with respect to the static magnetic field, of 21 +/- 2 degrees and, at the same time, map the rotation angle of the transmembrane domain with respect to the bilayer. PISEMA spectra obtained with selectively labeled samples show that the cytoplasmic domain of PLN is helical and makes an angle of 93 +/- 6 degrees with respect to the bilayer normal. In addition, using samples tilted by 90 degrees , we find that the transmembrane domain of PLN undergoes fast long-axial rotational diffusion about the bilayer normal with the cytoplasmic domain undergoing this motion and other complex dynamics, scaling the values of chemical shift anisotropy. While this dynamic was anticipated by previous solution NMR relaxation studies in micelles, these measurements in the anisotropic lipid environment reveal new dynamic and conformational features encoded in the free protein that might be crucial for SERCA recognition and subsequent inhibition.

A large volume flat coil probe for oriented membrane proteins

15N detection of mechanically aligned membrane proteins benefits from large sample volumes that compensate for the low sensitivity of the observe nuclei, dilute sample preparation, and for the poor filling factor arising from the presence of alignment plates. Use of larger multi-tuned solenoids, however, is limited by wavelength effects that lead to inhomogeneous RF fields across the sample, complicating cross-polarization experiments. We describe a 600MHz 15N–1H solid-state NMR probe with large (580mm3) RF solenoid for high-power, multi-pulse sequence experiments, such as polarization inversion spin exchange at the magic angle (PISEMA). In order to provide efficient detection for 15N, a 4-turn solenoidal sample coil is used that exceeds 0.27λ at the 600MHz 1H resonance. A balanced tuning-matching circuit is employed to preserve RF homogeneity across the sample for adequate magnetization transfer from 1H to 15N. We describe a procedure for optimization of the shorted 1/4λ coaxial trap that allows for the sufficiently strong RF fields in both 1H and 15N channels to be achieved within the power limits of 300W 1H and 1kW 15N amplifiers. The 8×6×12mm solenoid sustains simultaneous B1 irradiation of 100kHz at 1H frequency and 51kHz at 15N frequency for at least 5ms with 265 and 700W of input power in the respective channels. The probe functionality is demonstrated by 2D 15N–1H PISEMA spectroscopy for two applications at 600MHz.

Broadband-PISEMA solid-state NMR spectroscopy

The measurement of heteronuclear dipolar couplings using a 2D separated-local-field technique, Polarization Inversion Spin Exchange at the Magic Angle (PISEMA), is a unique way to determine the structure, dynamics and topology of molecules in solid-state. However, the resolution and sensitivity of PISEMA are highly dependent on the offset frequency of protons. To overcome this difficulty, in this study, a broadband-PISEMA pulse sequence is proposed. Experimental data from a single crystal and simulated results suggest that the new sequence compensates the offset effects. This is accomplished using a pair of 180° pulses that invert the spin-locked magnetization of I and S nuclei after certain number of SEMA cycles in the t 1 period. In addition, unlike PISEMA, BB-PISEMA provides offset-independent dipolar coupling line shapes even when low rf fields are applied.

Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy

We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and 1H– 15N heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [ 15N]Met-bR revealed clearly distinguishable signals from the helical and loop regions. By deconvolution of the helix resonances, it was possible to establish constraints for some helix tilt angles. It was estimated that the extracellular section of helix B has a tilt of less than 5° from the membrane normal, while the tilt of helix A was estimated to be 18–22°, both of which are in agreement with most crystal structures. Comparison of the experimental PISEMA spectrum with simulated spectra based on crystal structures showed that PISEMA and HETCOR experiments are extremely sensitive to the polytopic protein structure, and the solid-state NMR spectra for membrane-embedded bR matched most favorably with the recent 1FBB electron crystallography structure. These results suggest that this approach has the potential to yield structural and orientational constraints for large integral polytopic proteins whilst intercalated and functionally competent in a natural membrane.

A “Magic Sandwich” pulse sequence with reduced offset dependence for high-resolution separated local field spectroscopy

A pulse sequence for high resolution separated local field spectroscopy based on “magic sandwich” elements is demonstrated on a single crystal sample. Simulations and experimental results show that this pulse sequence has a reduced frequency offset dependence compared to PISEMA (polarization inversion spin exchange at the magic angle). As a result, it has a larger effective range of homonuclear decoupling, reduced zero-frequency spectral distortions, and more reliable scale factors for individual resonances. In addition, it is easier to setup on commercial spectrometers.

Separated local field spectroscopy of columnar and nematic liquid crystals

We are in this work comparing the efficiencies of various 1 H– 13 C separated local field (SLF) experiments when applied to columnar and nematic liquid crystals. In particular, the performances of the conventional SLF, proton-detected local field (PDLF), and polarization inversion spin exchange at the magic angle (PISEMA) methods in terms of spectral resolution, robustness, and ability to measure long-range couplings are investigated. The PDLF sequence provides in most cases the best dipolar resolution. This is especially obvious for weakly coupled 1 H– 13 C spin pairs.

Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints.

A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor "shotgun," of (15)N-labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not. Shotgun NMR short-circuits the laborious and time-consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins. As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination. A total of five two-dimensional (1)H/(15)N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively (15)N-labeled samples, were sufficient to determine the structure of the membrane-bound form of the 50-residue major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three-dimensional structure of the protein with atomic resolution. The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy. [The structure we determined for the membrane-bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.]

The effect of Hartmann–Hahn mismatching on polarization inversion spin exchange at the magic angle

The effect of the Hartmann–Hahn mismatch Δ= ω eff− ω 1S during polarization inversion spin exchange at the magic angle (PISEMA) has been investigated, where ω eff and ω 1S represent the amplitudes of the 1H effective spin-locking field at the magic angle and the 15N RF spin-locking field, respectively. During the PISEMA evolution period, the exact Hartmann–Hahn match condition (i.e., Δ=0) yields a maximum dipolar scaling factor of 0.816 for PISEMA experiments, while any mismatch results in two different effective fields for the first and second half of each frequency switched Lee–Goldburg (FSLG) cycle. The mismatch effect on the scaling factor depends strongly on the transition angle from one effective field to the other within each FSLG cycle as well as on the cycle time. At low RF spin-lock amplitudes in which the FSLG cycle time is relatively long, the scaling factor rapidly becomes smaller as ω 1S becomes greater than ω eff. On the other hand, when ω 1S< ω eff, there is relatively little effect on the scaling factor with variation in Δ. As a result, the presence of RF inhomogeneities may significantly broaden the line-width in the dipolar dimension because of the mismatch effect. Higher RF spin-lock amplitudes result in a relatively small variation for the scaling factor. Furthermore, ramped amplitude of the 15N RF spin-lock field in synchronization with the flip-flop of the FSLG sequence minimizes the transition angle between the two effective fields within the FSLG cycle. It is shown experimentally that such a ramped amplitude not only gives rise to the same scaling factor but also results in a narrower dipolar line-width in comparison with the rectangular amplitude.

Structure determination of membrane proteins by NMR spectroscopy

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.

Dipolar waves as NMR maps of protein structure.

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.

Spin Dynamics of Polarization Inversion Spin Exchange at the Magic Angle in Multiple Spin Systems

Polarization inversion spin exchange at the magic angle (PISEMA) [J. Magn. Reson. A 109, 270 (1994)] is an important experiment in NMR structural characterization of membrane proteins in oriented lipid bilayers. This paper presents a theoretical and experimental study of the spin dynamics in PISEMA to investigate the line-narrowing mechanism. The study focuses on the effect of neighboring protons on the spin exchange of a strongly coupled spin pair. The spin exchange is solved analytically for simple spin systems and is numerically simulated for many-spin systems. The results show that the dipolar couplings from the neighboring protons of a strongly coupled spin pair perturb the spin exchange only in the second order, therefore it has little contribution to the linewidth of PISEMA spectra in comparison to the separated-local-field spectra. The effects from proton resonance offset and the mismatch of the Hartmann–Hahn condition are also discussed along with experimental results using model single-crystal samples.