¹⁵N T₂′ relaxation times of bacteriorhodopsin transmembrane amide nitrogens

Abstract

15N T(2)' relaxation times of bacteriorhodopsin (BR) amide nitrogens were determined in the temperature range from 40 to -60 degrees C using a Hahn echo pulse sequence and proton decoupling during the echo and detection times. Using oriented membrane samples, with their bilayer normal parallel to the external magnetic field, the (15)N amide nitrogens belonging to the transmembrane helices could be selected for the analysis. The experiments were performed on purple membrane fragments (in which BR is organized in a 2D crystalline network) and on BR reconstituted into dimyristoylphosphatidylcholine at a 1:150 molar ratio (in which BR is in a freely diffusing monomeric state at 40 degrees C and in an aggregated state at 4 degrees C and below). The results are discussed in terms of helix dynamics, mosaic spread and resolution of the (15)N spectra for the various samples and experimental conditions.

Heteronuclear chemical shift correlation and J‐resolved MAS NMR spectroscopy of lipid membranes

“…The fused‐ring structures of cholesterol rigidify the molecule, which leads to restricted lateral excursions of lipid chain methylenes near the glycerol backbone and increase in their orientational order. Heteronuclear chemical shift correlation spectroscopy has been used to obtain J ‐couplings from sterols using MAS‐J‐HMQC,45 while a substantial body of work has been carried out using MAS‐INEPT26 and HSQC36 to study membrane cholesterol and HETCOR to investigate ergosterol/phospholipid systems 27. Here, we report results from a study of hydrated membranes composed of DOPC/cholesterol in a 2/1 mixture, using undecoupled evolution 1 H 13 C HETCOR with SPINAL or FSLG proton decoupling during carbon acquisition (Fig.…”

“…Lipid membranes composed of PC, cholesterol and sphingomyelin can form laterally separated ordered phases co‐existing with less ordered lateral environments 48, 49. Sterol‐containing membranes have been studied by HSQC‐MAS26, 36 and heteronuclear correlation spectroscopy with INEPT transfer 24. Sphingomyelin–cholesterol bilayers have been investigated by FSLG‐HETCOR23 and spectra have been acquired both from fluid membranes and from bilayers in the gel phase.…”

“…The effects of cholesterol on membrane lipids have been investigated by NOESY‐MAS 15. Chemical shift‐correlated spectra from sterol‐containing membranes have been obtained using refocused wide line separation (WISE)‐type experiments with or without MREV‐8 homonuclear decoupling during evolution, followed by CP or INEPT transfer to 13 C 26, 27. Under free evolution, Hong et al 28, 29 have observed WISE‐type heteronuclear chemical shift correlation spectra, which show J ‐resolved proton doublets.…”

Solid-State NMR Spectroscopy of Oriented Membrane Polypeptides at 100 K with Signal Enhancement by Dynamic Nuclear Polarization

“…Solid-state NMR spectroscopy provides a powerful tool for the structural investigation of membrane peptides and proteins in their native lipid environment. – Polypeptides reconstituted in membranes have been studied by magic angle spinning approaches which result in spectra that resemble NMR spectra in solution and which provide chemical shifts and distance correlations that are used for structural analysis. Alternatively, static solid-state NMR approaches have been developed for oriented – or nonoriented samples , from which angular constraints are obtained for structural analysis. Whereas solid-state NMR spectroscopy has been used to study the structure, dynamics, and topology of these proteins it has been difficult to develop its full potential due to the inherently low sensitivity of NMR spectroscopy which is associated with the small energy difference between the nuclear Zeeman eigenstates.…”

Side Chain Resonances in Static Oriented Proton-Decoupled 15N Solid-State NMR Spectra of Membrane Proteins

“…Solid-state NMR spectroscopy is a powerful technique to investigate the structure, topology, and dynamics of proteins in microcrystals − or when reconstituted into membranes. − On the one hand magic angle spinning multidimensional high-resolution solid-state NMR spectra are obtained from proteins labeled with 15 N and 13 C provided that the sample is characterized by a high local order. The correlations that are detected in these spectra provide valuable distance and chemical shift information that can be used for structural analysis.…”