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

Abstract

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...

Complete resolution of the solid-state NMR spectrum of a uniformly 15 N-labeled membrane protein in phospholipid bilayers

“…2 (25,39,40). Also, any magnetization transferred from the 1 H spin reservoir to the 15 N spin reservoir through cross-polarization before polarization inversion, which does not participate in spin exchange at the magic angle, appears at zero dipolar frequency.…”

“…The stack of glass plates was placed in a rectangular, five-turn coil with inner dimensions of 20 ϫ 11 ϫ 5 mm. This high power ''square'' coil version of the original flat coil probe (34) was shown previously to have adequate rf homogeneity for the homonuclear line-narrowing procedures used here (25). The unoriented sample was placed in a 7-mm solenoidal coil.…”

“…We have recently developed a family of two-, three-, and four-dimensional solid-state NMR experiments (21-24) for high resolution spectroscopy and structure determination of proteins (25,26). Here we demonstrate that a fully resolved three-dimensional solid-state NMR correlation spectrum can be obtained from a uniformly 15 N-labeled membrane protein in phospholipid bilayers.…”

The Solution Structure and Activity of Caerin 1.1, an Antimicrobial Peptide from the Australian Green Tree Frog, Litoria Splendida

“…The amphipathic helical conformation of caerin 1.1 determined herein, therefore, may be the one that the peptide adopts when interacting with membranes. Indeed for magainin, solidstate NMR studies of the peptide incorporated into lipid bilayers show that it adopts a helical conformation aligned parallel to the plane of the membrane (Bechinger et al, 1991(Bechinger et al, , 1993Ramamoorthy et al, 1995). Fig.…”

Structure, Topology, and Tilt of Cell-Signaling Peptides Containing Nuclear Localization Sequences in Membrane Bilayers Determined by Solid-State NMR and Molecular Dynamics Simulation Studies

“…Since most of the 15 N labeled residues are most likely located in the hydrophobic regions of the bilayer, providing considerably less conformational or dynamic disorder, narrow lines are observed in the spectra of aligned samples. On the other hand, some amphipathic peptides oriented near the surface of bilayers could result in broad lines due to heterogeneous orientation (12,15,58) or narrow lines due to motion (59). The observed line widths range from 1.8 to 6 ppm and did not depend on the RF power of proton decoupling.…”