Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides

Abstract

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.

Reluctance to membrane binding enables accessibility of the synaptobrevin SNARE motif for SNARE complex formation

“…Application of solution NMR methods to study the structure of membrane proteins reconstituted into phosphoplipid vesicles is hindered by the very large size of the vesicles, which effectively ranges from tens of megadaltons to gigadaltons depending on their radii and leads to broadening beyond detection of resonances from structured parts of the proteins. This problem can be alleviated by incorporation of membrane proteins into nanodiscs (30)(31)(32), which leads to effective molecular weights in the 150-400 kDa range but still requires protein perdeuteration to obtain high-quality NMR data for structured regions. In the current study, we made no attempt to observe resonances from structured regions of reconstituted synaptobrevin and focused on determining which regions of its sequence remain flexible upon anchoring to a phospholipid bilayer.…”

Functional Equilibrium of the KcsA Structure Revealed by NMR

“…KcsA125 was obtained by cleaving the C-terminal intracellular region of wild-type KcsA by chymotrypsin. The plasmid for the expression of the membrane scaffold protein MSP1 was constructed according to the literature (17)(18)(19). The BL21 (DE3) Codon Plus RP Escherichia coli cells were transformed with the plasmid and cultured in Terrific Broth medium at 37°C.…”

“…Reconstitution of KcsA in rHDL-Reconstitution of KcsA into rHDL was conducted according to a previous report (19), with some modifications. Delipidations of MSP1 and KcsA were confirmed by 31 P 1D NMR spectroscopy.…”

“…The film was solubilized with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 150 mM KCl, and 50 mM sodium cholate. After sonication, MSP1 was added to a final concentration of 200 M. After an incubation for 2 h at room temperature with gentle mixing, KcsA was added to achieve a final molar ratio of KcsA, MSP1, lipids, and sodium cholate of 1:20:800:1600 (19). After incubating the solution at room temperature for 2 h, 50% (w/w) of Biobeads (Bio-Rad), which adsorb detergents, was added in a stepwise manner, and the mixture was incubated at room temperature overnight with gentle mixing.…”

“…detergent micelles or in the lipid bilayer (23). We then investigated the p perm values in the lipid bilayer under acidic conditions by embedding KcsA in rHDL, in which the KcsA reconstituted in the lipid bilayer is surrounded by membrane scaffold proteins to form a disc-shaped particle (17,19 S2). Compared with the spectra recorded in the DDM micelles, small but significant differences in the chemical shifts were observed, not only for the residues exposed to lipid bilayer in rHDL but also for the residue of the selectivity filter, Val-76, and those peripheral to the selectivity filter, Leu-59 and Leu-81 (supplemental Fig.…”