Improving Sensitivity in Mechanically Oriented Phospholipid Bilayers Using Ultrathin Glass Plates - A Deuterium Solid-State NMR Study

Prosser, R. Scott; Hunt, Sheri A.; Vold, Regitze R.

doi:10.1006/jmrb.1995.1156

Cited by 22 publications

(31 citation statements)

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“…The sample with two different labeled magainins was prepared by mixing 7 mg of each of the labeled peptides with sufficient palmitoyl-oleoylphosphatidylcholine (POPC) (80%) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) (20%) to obtain a final molar ratio of 4% peptide in the lipids. The mixtures of peptides and lipids were co-solubilized in chloroform with a trace amount of trifluoroethanol and then spread on the surface of 20 18×18 mm glass plates, the thickness of which was reduced from 0.1 to 0.06 mm by etching in a solution of 8% HF in ethanol (v/v) as described by Prosser et al (1995), to give the highest possible filling factor within the basic geometry of a fiat-coil probe. After air drying of the sample, residual solvent was removed under vacuum for 2 h. The plates were then stacked and the sample was hydrated at 45 °C for more than 12 h. After hydration, the sample was equilibrated at 93% relative humidity for an extended period of time in a closed chamber.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Ramamoorthy

Marassi

Zasloff³

et al. 1995

J Biomol NMR

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SummaryA three-dimensional 1 H chemical shift/ 1 H-15 N dipolar coupling/ 15 N chemical shift correlation spectrum was obtained on a sample of specifically 15 N-labeled magainin peptides oriented in lipid bilayers between glass plates in a flat-coil probe. The spectrum showed complete resolution of the resonances from two labeled amide sites in all three dimensions. The three orientationally dependent frequencies associated with each resonance enabled the orientation of the peptide planes to be determined relative to the direction of the applied magnetic field. These results demonstrate the feasibility of multiple-pulse spectroscopy in a flat-coil probe, the ability to measure three spectral parameters from each site in a single experiment, and the potential for resolving among many labeled sites in oriented membrane proteins. KeywordsSolid-state NMR; Magainin; Membranes; Oriented samples; Structure determination Solid-state NMR spectroscopy of macroscopically oriented systems is a particularly appealing approach to the structural characterization of peptides and proteins associated with bilayer membranes (Cross and Opella, 1994), since their three-dimensional structures are intimately related to the highly ordered and anisotropic environment of phospholipid bilayers, and suitable well-oriented samples can be prepared for these compounds. Orientationally dependent spectral parameters are measured and then interpreted in terms of the angles between the individual bonds and the direction of the applied magnetic field. Generally, this has been accomplished using specifically labeled samples, where the spectral parameters from one or, at most, a few sites are measured in each experiment . The implemenuation of three-and four-dimensional solid-state NMR experiments (Ramamoorthy et al., 1995a,b) in Structural studies of oriented membrane samples is attractive, because it gives opportunities for resolving among multiple overlapping resonances and for measuring multiple spectral parameters from experiments on uniformly labeled peptides and proteins.In this communication we describe the application of a recently developed threedimensional solid-state NMR experiment (Ramamoorthy et al., 1995a) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript resulting 3D spectrum is characterized by three frequencies that can be analyzed by the graphical restriction plot method to yield the orientations of the individual peptide planes, which can then be assembled to form the polypeptide structure . The spectrum also demonstrates the potential for resolution among resonances from multiple sites in uniformly labeled proteins. An important technical finding is that a flat-coil probe has adequate rf homogeneity for multiple-pulse line narrowing of 1 H resonances.The samples used for these studies consisted of specifically 15 N-labeled magainin2 peptides in hydrated phospholipid bilayers oriented between glass plates. The magainins are a family of 21-26-residue amphipathic helical peptides, found originally in frog...

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Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Ramamoorthy

Marassi

Zasloff³

et al. 1995

J Biomol NMR

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“…The spectrum obtained from an oriented sample displays significant resolution with identifiable peaks at frequencies throughout the range of the 15 N amide chemical shift anisotropy powder pattern. Residues in the transmembrane helix have N-H bonds oriented approximately parallel to the field and to the σ 33 component of the chemical shift tensor, while residues in the protein amphipathic in-plane helix have their N-H bonds perpendicular to the field and to σ 33 . 6,29 Two different methods for orienting lipid bilayers on glass plates are used, depending on the polypeptide, in order to obtain the best sample orientation.…”

Section: Glass-supported Lipid Bilayersmentioning

confidence: 99%

“…The thickness of the plates can be further reduced by etching with HF. 33 Deposition from organic solvents: This method works well for the reconstitution and orientation of relatively small polypeptides, and we have prepared highly oriented samples of magainin and M2 peptides from acetylcholine receptor (AchR) and the NMDA receptor (NMDAR) in this way. For the AChR M2 samples, 40 mg of 1,2-dimyristoyl-snglycerophosphocholine (DMPC) powder (Avanti Polar Lipids, Alabaster, AL) is dissolved in trifluoroethanol (TFE) and added to 2 mg of lyophilized, HPLC purified peptide.…”

Section: Glass-supported Lipid Bilayersmentioning

confidence: 99%

“…Void and co-workers 33 demonstrated that the addition of a small amount of lanthanide ions "flips" the bicelles to the parallel orientation. Subsequently this has become an active area of research in a number of different laboratories.…”

Section: Magnetically Oriented Lipid Bicellesmentioning

confidence: 99%

“…Many transmembrane helices are tilted with respect to the bilayer normal. The combination of tilt and the 17° angle between the σ 33 and NH bond vectors makes it possible to resolve many resonances from residues in otherwise uniform helices and is responsible for the "wheel-like" pattern observed in two-dimensional PISEMA spectra of uniformly 15 N labeled proteins.…”

Section: Structure Determination Of Membrane Proteins In Lipid Bilayersmentioning

confidence: 99%

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Improving Sensitivity in Mechanically Oriented Phospholipid Bilayers Using Ultrathin Glass Plates - A Deuterium Solid-State NMR Study

Cited by 22 publications

References 0 publications

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Nuclear Magnetic Resonance of Membrane-Associated Peptides and Proteins

Lanthanide ion assisted magnetic alignment of model membranes and macromolecules

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