Polarization Inversion Spin Exchange At Magic Angle Research Articles

Simulation of oriented NMR spectra: Combining molecular dynamics and chemical shift tensor calculations.

Solid state NMR is widely used to study the orientation and other structural features of proteins and peptides in lipid bilayers. Using data obtained by PISEMA (Polarization Inversion Spin Exchange at Magic Angle) experiments, periodic spectral patterns arise from well-aligned α-helical molecules. Significant problems in the interpretation of PISEMA spectra may arise for systems that do not form perfectly defined secondary structures, like α-helices, or the signal pattern is disturbed by molecular motion. Here, we present a new method that combines molecular dynamics simulation with tensorial orientational constraints (MDOC) and chemical shift tensor calculations for the simulation and interpretation of PISEMA-like spectra. The calculations include the spectra arising from non α-helical molecules and molecules with non-uniform intrinsic mobility. In a first step, dipolar or quadrupolar interaction tensors drive molecular rotations and reorientations to obtain the proper mean values as observed in corresponding NMR experiments. In a second step, the coordinate snapshots of the MDOC simulations are geometry optimized with the isotropic 15 N chemical shifts as constraints using Bond Polarization Theory (BPT) to provide reliable 15 N CS tensor data. The averaged dipolar 1 H-15 N couplings and the δzz tensor components can then be combined to simulate PISEMA patterns. We apply this method to the ß-helical peptide gramicidin A (gA) and demonstrate that this method enables the assignment of most PISEMA resonances. In addition, MDOC simulations provide local order parameters for the calculated sites. These local order parameters reveal large differences in backbone mobility between L- and D-amino acids of gA.

Effect of PEO molecular weight on the miscibility and dynamics in epoxy/PEO blends.

In this work, the effect of poly(ethylene oxide) (PEO) molecular weight in blends of epoxy (ER) and PEO on the miscibility, inter-chain weak interactions and local dynamics were systematically investigated by multi-frequency temperature modulation DSC and solid-state NMR techniques. We found that the molecular weight (M(w)) of PEO was a crucial factor in controlling the miscibility, chain dynamics and hydrogen bonding interactions between PEO and ER. A critical PEO molecular weight (M(crit)) around 4.5k was found. PEO was well miscible with ER when the molecular weight was below M(crit), where the chain motion of PEO was restricted due to strong inter-chain hydrogen bonding interactions. However, for the blends with high molecular weight PEO (M(w) > M(crit)), the miscibility between PEO and ER was poor, and most of PEO chains were considerably mobile. Finally, polarization inversion spin exchange at magic angle (PISEMA) solid-state NMR experiment further revealed the different mobility of the PEO in ER/PEO blends with different molecular weight of PEO at molecular level. Based on the DSC and NMR results, a tentative model was proposed to illustrate the miscibility in ER/PEO blends.

Insights into the Molecular Dynamics in Polysulfone Polymers from 13C Solid-State NMR Experiments

The molecular and segmental motions in three different grades of ductile polysulfone polymers; poly(ether sulfone) (PESU), polysulfone (PSU), and poly(phenyl sulfone) (PPSU) are probed using (13)C solid-state NMR experiments. Polarization inversion spin exchange at magic angle (PISEMA) experiments indicates that the phenyl rings in the polymers are undergoing π-flip motions on the order of 100 kHz. The temperature dependent PISEMA experiments show that the fraction of mobile regions that undergoes aromatic π-flips is higher in PPSU than in the other two polymers. The center band only detection of exchange (CODEX) experiments was carried out and was unable to detect any slow segmental motions in the chains. A combination of (13)C spin-lattice relaxation time (T1) and T1-filtered PISEMA experiments show that the mobile regions in all the polymers are dynamically heterogeneous.

Structure-Function Studies of Mtb Membrane Protein CrgA in Lipid Bilayer

Tuberculosis is a deadly disease with a death toll of 1.5 million people every year and very recently an outbreak in Jacksonville, FL was devastating. All frontline antibiotics are failed for the multidrug resistant (MDR) bacilli and the key to disease control is to inhibit bacterial cell division and stabilize the bacilli in its dormant stage. Here we present the structure function studies of a novel drug target membrane protein CrgA in M. tuberculosis peptidoglycan polymerization complex. We show that CrgA interacts strongly with FtsZ, FtsQ, CwsA and the class B penicillin binding proteins such as FtsI and PBPA in M. smegmatis. CwsA a binding partner of CrgA binds to Wag31 another key protein in peptidoglycan synthesis. Co-localization of CrgA with FtsZ in the mid cell region facilitates the Z-ring formation during the cell division process. Thus structural characterization of CrgA in a lipid bilayer is essential. Full length CrgA has 2 transmembrane (TM) helices, a 32 residue N terminal domain, a very short C terminal domain, and a 10 residue inter-helical loop for a total of 93 residues and an 11.4 kDa molecular weight. Full length 15N, 13C uniform and amino acid specific labeled CrgA was over-expressed and purified from E. coli with a yield of 35 mg/L. To mimic the membrane environment, isotope labeled CrgA was reconstituted in POPC:POPG liposomes. Orientation dependent 2D Polarization Inversion Spin Exchange at Magic Angle (PISEMA) and 3D Magic Angle Spinning (MAS) solid state NMR experiments were performed to obtain structural restraints. For all ssNMR experiments Low E probes in 600 and 900 MHz magnets were used at the NHMFL. CrgA TMD structure was calculated by CS-Rosetta and XPLOR-NIH. Structure refinement was enhanced by restrained molecular dynamics in a lipid bilayer environment.

Comparative analysis of the orientation of transmembrane peptides using solid-state 2H- and 15N-NMR: mobility matters

Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters, from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches: (2)H-NMR quadrupolar waves, and separated local field (15)N-(1)H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational fluctuations, which are not directly seen by (2)H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23, though the helix tilt could still be determined, irrespective of any dynamics parameters.

Accessing Structure and Dynamics of Mobile Phase in Organic Solids by Real-Time T1C Filter PISEMA NMR Spectroscopy

The structure and dynamic behavior of mobile components play a significant role in determining properties of solid materials. Herein, we propose a novel real-time spectrum-editing method to extract signals of mobile components in organic solids on the basis of the polarization inversion spin exchange at magic angle (PISEMA) pulse sequence and the difference in (13)C T(1) values of rigid and mobile components. From the dipolar splitting spectrum sliced along the heteronuclear dipolar coupling dimension of the 2D spectrum, the structural and dynamic information can be obtained, such as the distances between atoms, the dipolar coupling strength, the order parameter of the polymer backbone chain, and so on. Furthermore, our proposed method can be used to achieve the separation of overlapped NMR signals of mobile and rigid phases in the PISEMA experiment. The high efficacy of this 2D NMR method is demonstrated on organic solids, including crystalline L-alanine, semicrystalline polyamide-6, and the natural abundant silk fibroin.

Efficient Identification of Different Types of Carbons in Organic Solids by 2D Solid-State NMR Spectroscopy

An efficient method for identifying different types of carbon groups (CH(3), CH(2), CH, and quaternary carbons) in organic solids is proposed by utilizing the combination of a two-dimensional (2D) (13)C-(1)H polarization inversion spin exchange at magic angle (PISEMA) NMR experiment and numerical simulation results of simple isolated (13)C-(1)H dipolar coupling models. Our results reveal that there is a unique line shape of the (13)C-(1)H dipolar splitting pattern and a corresponding characteristic splitting value for each carbon group, based on which different carbon types can be distinguished unambiguously. In particular, by using this method, the discrimination and assignment of overlapped signals from different types of carbons can be achieved easily. The efficacy of this method is demonstrated on typical solid small molecules, polymers, and biomacromolecules.

2 4-SEMA as a sensitive and offset compensated SLF sequence

Separated Local Field (SLF) spectroscopy is a powerful tool for the determination of structure and dynamics of oriented systems such as membrane proteins oriented in lipid bilayers and liquid crystals. Of many SLF techniques available, Polarization Inversion Spin Exchange at Magic Angle (PISEMA) has found wide application due to its many favorable characteristics. However the pulse sequence suffers from its sensitivity to proton resonance frequency offset. Recently we have proposed a new sequence named 2 4-SEMA (J. Chem. Phys. 132 (2010) 134301) that overcomes this problem of PISEMA. The present work demonstrates the advantage of 2 4-SEMA as a highly sensitive SLF technique even for very large proton offset. 2 4-SEMA has been designed for obtaining reliable dipolar couplings by switching the magic-angle spin-lock for protons over four quadrants as against the use of only two quadrants in PISEMA. It is observed that for on-resonance condition, 2 4-SEMA gives rise to signal intensity comparable to or slightly higher than that from PISEMA. But under off-resonance conditions, intensities from 2 4-SEMA are several fold higher than those from PISEMA. Comparison with another offset compensated pulse sequence, SAMPI4, also indicates a better intensity profile for 2 4-SEMA. Experiments carried out on a single crystal of 15N labeled N-acetyl– dl-valine and simulations have been used to study the relative performance of the pulse sequences considered.

2 n -SEMA -a robust solid state nuclear magnetic resonance experiment for measuring heteronuclear dipolar couplings in static oriented systems using effective transverse spin-lock

Separated local field (SLF) spectroscopy is a powerful technique to measure heteronuclear dipolar couplings. The method provides site-specific dipolar couplings for oriented samples such as membrane proteins oriented in lipid bilayers and liquid crystals. A majority of the SLF techniques utilize the well-known Polarization Inversion Spin Exchange at Magic Angle (PISEMA) pulse scheme which employs spin exchange at the magic angle under Hartmann-Hahn match. Though PISEMA provides a relatively large scaling factor for the heteronuclear dipolar coupling and a better resolution along the dipolar dimension, it has a few shortcomings. One of the major problems with PISEMA is that the sequence is very much sensitive to proton carrier offset and the measured dipolar coupling changes dramatically with the change in the carrier frequency. The study presented here focuses on modified PISEMA sequences which are relatively insensitive to proton offsets over a large range. In the proposed sequences, the proton magnetization is cycled through two quadrants while the effective field is cycled through either two or four quadrants. The modified sequences have been named as 2(n)-SEMA where n represents the number of quadrants the effective field is cycled through. Experiments carried out on a liquid crystal and a single crystal of a model peptide demonstrate the usefulness of the modified sequences. A systematic study under various offsets and Hartmann-Hahn mismatch conditions has been carried out and the performance is compared with PISEMA under similar conditions.

Sensitivity enhancement in static solid-state NMR experiments via single- and multiple-quantum dipolar coherences.

We present a new method for enhancing the sensitivity in static solid-state NMR experiments for a gain in signal-to-noise ratio of up to 40%. This sensitivity enhancement is different from the corresponding solution NMR sensitivity enhancement schemes and is achieved by combining single- and multiple-quantum dipolar coherences. While this new approach is demonstrated for the polarization inversion spin exchange at magic angle (PISEMA) experiment, it can be generalized to the other separated local field experiments for solid-state NMR spectroscopy. This method will have a direct impact on solid-state NMR spectroscopy of liquid crystals as well as of membrane proteins aligned in lipid membranes.

14N Polarization Inversion Spin Exchange at Magic Angle (PISEMA)

Polarization Inversion Spin Exchange at Magic Angle (PISEMA) is a powerful experiment for determining peptide orientation in uniformly aligned samples such as planar membranes. In this paper, we present 14N-PISEMA experiment which correlates 14N quadrupolar coupling and 14N– 1H dipolar coupling. 14N-PISEMA enables the use of 14N quadrupolar coupling tensor as an ultra sensitive probe for peptide orientation and can be carried out without the need of isotope enrichment. The experiment is based on selective spin-exchange between a proton and a single-quantum transition of 14N spins. The spin-exchange dynamics is described and the experiment is demonstrated with a natural abundant N-acetyl valine crystal sample.

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.

Transmembrane Domain of M2 Protein from Influenza A Virus Studied by Solid-State 15N Polarization Inversion Spin Exchange at Magic Angle NMR

The M2 protein from the influenza A virus forms a proton channel in the virion that is essential for infection. This tetrameric protein appears to form a four-helix bundle spanning the viral membrane. Here the solid-state NMR method, 2D polarization inversion spin exchange at magic angle (PISEMA), has been used to obtain multiple constraints from specifically amino acid-labeled samples. The improvement of spectral resolution from 2D PISEMA over 1D methods and 2D separated local field methods is substantial. The reliability of the method is validated by comparison of anisotropic chemical shift and heteronuclear dipolar interactions from single site labeled samples. The quantitative interpretation of the high-resolution constraints confirms the helix tilt to be within the range of previous experimental determinations (32°–38°). The binding of the channel inhibitor, amantadine, results in no change in the backbone structure at position Val 27,28, which is thought to be a potential binding site for the inhibitor.