9EMZ

13C/15N-labelled Integral Membrane Enzyme LspA in the Lipid Cubic Phase

9EMZ の概要

• エントリーDOI: 10.2210/pdb9emz/pdb
• 関連するPDBエントリー: 5DIR
• 分子名称: Lipoprotein signal peptidase, Globomycin, (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate, ... (4 entities in total)
• 機能のキーワード: lipid cubic phase, lspa, hydrolase, globomycin
• 由来する生物種: Pseudomonas aeruginosa PAO1; 詳細
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 87305.24

構造登録者

Bailey, J.,Boland, C.,Caffrey, M.,Huang, C.-Y.,Gawrisch, K.,Kooshapur, H.,Ramberg, K.O.,Soubias, O.,Wiktor, M. (登録日: 2024-03-12, 公開日: 2025-03-26, 最終更新日: 2025-05-21)

主引用文献

Ramberg, K.O.,Boland, C.,Kooshapur, H.,Soubias, O.,Wiktor, M.,Huang, C.Y.,Bailey, J.,Gawrisch, K.,Caffrey, M.
Solid-state NMR of membrane peptides and proteins in the lipid cubic phase.
Biophys.J., 124:1387-1400, 2025

PubMed Abstract: Solid-state nuclear magnetic resonance (ssNMR) is a powerful technique for studying membrane protein structure and dynamics. Ideally, measurements are performed with the protein in a lipid bilayer. However, homogenous reconstitution of functional protein into intact bilayers at sufficiently high concentrations is often difficult to achieve. In this work, we investigate the suitability of the lipid cubic phase (LCP), which incorporates a lipid bilayer, as an alternative medium for ssNMR of integral membrane peptides and proteins. The cubic mesophase has long been used to generate membrane protein crystals for use in X-ray crystallographic structure determination by the so-called in meso method and for protein functional and biophysical characterization. Preparing and handling protein-laden LCP is straightforward. LCP may therefore provide a valuable alternative to native membranes and other membrane mimetics for ssNMR. We tested this idea by conducting standard magic-angle spinning ssNMR experiments on LCP into which gramicidin, a ∼4-kDa transmembrane peptide, or bacterial lipoprotein signal peptidase II (LspA), a ∼20-kDa integral membrane enzyme, had been reconstituted. We report one- and two-dimensional ssNMR spectra for both gramicidin and LspA and the parameters for optimizing spectral quality. The high protein-carrying capacity of the cubic phase facilitated C ssNMR at natural abundance. Lowering temperature and raising magic-angle spinning frequency enabled significant improvements in spectral quality. One-dimensional C and N spectra were collected for LspA. Two-dimensional ssNMR experiments provided information on LspA dynamics and its interaction with the water and lipid components of the cubic phase. Solution NMR measurements carried out in parallel yielded information on the effect of the antibiotic, globomycin, on LspA structure and dynamics.

PubMed: 40119522
DOI: 10.1016/j.bpj.2025.03.012

実験手法

X-RAY DIFFRACTION (3 Å)