In Situ Structure Determination of a Membrane Protein in Native Cellular Membranes by Proton-Detected Solid-State NMR

Determining the structure of membrane proteins within their native cellular membranes remains a substantial challenge in structural biology. In this study, we present a proton-detected solid-state NMR (ssNMR) approach, combined with an optimized random partial protonation (RAP) labeling strategy, to determine the high-resolution structure of the large-conductance mechanosensitive channel (MscL) directly within native E.coli membranes (backbone RMSD = 1.9 [A]). Our approach effectively suppresses background protein signals and achieves high spectral resolution and sensitivity at moderate MAS frequencies (40-60 kHz) by differentially tuning amide and side-chain protonation levels. Using advanced recoupling schemes, we obtained chemical shift assignments of side-chain protons by 3D hCCH spectra and 1H-1H distance restraints from a series of 3D hNHH spectra. With 10% protonation in side-chains, the 1H signals displayed linewidths of approximately 50Hz, facilitating the extraction of 49 long-range distance restraints between amide and side-chain protons, which are crucial for structural convergence. Ambiguities in the assignment of weak signals corresponding to distance restraints were resolved by integrating 3D hNHH experimental data with CS-Rosetta structural modeling. The resulting structure reveals a well-defined pentameric assembly, with transmembrane helix packing consistent with that observed in detergent environments. This study demonstrates significant sensitivity advantages of 1H-detected over 13C-detected in situ ssNMR methods, highlighting the potential of 1H-detected ssNMR for the structure determination of a broad range of membrane proteins in native membranes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/685061v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1c2638dorg.highwire.dtl.DTLVardef@115e4d6org.highwire.dtl.DTLVardef@62c20forg.highwire.dtl.DTLVardef@1b29f67_HPS_FORMAT_FIGEXP M_FIG C_FIG