Conformational heterogeneity of transmembrane residues after the Schiff base reprotonation of bacteriorhodopsin

Abstract

bR, N‐like and O‐like intermediate states of [15N]methionine‐labelled wild type and D85N/T170C bacteriorhodopsin were accumulated in native membranes by controlling the pH of the preparations. 15N cross polarization and magic angle sample spinning (CPMAS) NMR spectroscopy allowed resolution of seven out of nine resonances in the bR‐state. It was possible to assign some of the observed resonances by using 13C/15N rotational echo double resonance (REDOR) NMR and Mn2+ quenching as well as D2O exchange, which helps to identify conformational changes after the bacteriorhodopsin Schiff base reprotonation. The significant differences in chemical shifts and linewidths detected for some of the resonances in N‐ and O‐like samples indicate changes in conformation, structural heterogeneity or altered molecular dynamics in parts of the protein.

How to Prepare Membrane Proteins for Solid‐State NMR: A Case Study on the α‐Helical Integral Membrane Protein Diacylglycerol Kinase from E. coli

“…[29] This labelling scheme results in fewer resonances and hence in an increase in resolution. This is due to a reduction in both the number of overlapping resonances and homonuclear 13 CÀ 13 C homogeneous line-broadening. [11,29,30] We demonstrate that SE-labelled, nondiffracting, crystalline protein can be used to acquire high-quality spectra with linewidths that are comparable to previously published SSNMR spectra of diffracting crystals.…”

“…However, reconstitution is the method of choice for biophysical studies by solid-state NMR. In addition, we discuss the identification of lipids bound to membrane-protein crystals by ples offer the option of investigating the dynamics of the protein as it binds ligands, [12] negotiates its reaction cycle [13] or responds to changes in the lipid environment. [14] SSNMR imposes some strict constraints on sample preparation.…”

“…Furthermore, we have prepared selectively and extensively (SE) labelled protein for 13 C-CPMAS and double-quantum-filtered experiments by using [2-13 C] glycerol as the sole carbon source. [29] This labelling scheme results in fewer resonances and hence in an increase in resolution.…”

“…For this case study, we have prepared proteoliposomes and small crystals of the a-helical membrane-protein diacylglycerol kinase (DGK). Preparations were characterised by 13 C-and 15 N-cross-polarization magic-angle spinning (CPMAS) NMR. It was found that crystalline samples produce better-resolved spectra than proteoliposomes.…”

Investigating transport proteins by solid state NMR

“…The latter is clearly the preparation of choice as it is closest to the protein's native environment. Lipid reconstituted samples also offer the option of investigating the dynamics of the protein as it binds ligands (Patching et al 2004a), negotiates its reaction cycle (Mason et al 2005) or responds to changes in the lipid environment (Yamaguchi et al 2004). However, the currently very limited knowledge base does not yet allow us to derive general rules about how to prepare membrane proteins for solid state NMR and so screens have to be performed for each individual protein.…”