Site-Directed Solid-State NMR on Membrane Proteins

Abstract

Well-resolved, fully visible 13C NMR signals of membrane proteins were successfully recorded either at ambient or at lower temperatures when they were embedded in lipid bilayers constituting a 2D crystalline lattice, as manifested from site-directed 13C NMR studies on [3-13C]Ala and/or [1-13C]Val-labeled bacteriorhodopsin (bR) from purple membrane, instead of uniformly labeled preparations. It is emphasized that recording 13C NMR spectra by dipolar decoupled magic angle spinning (DD-MAS) is essential to detect signals from rather flexible portions of fully hydrated membrane proteins, in addition to recording signals from static portions by cross-polarization-magic angle spinning (CP-MAS). The resulting 13C NMR signals were site-directly assigned to specific residues utilizing site-directed mutants, otherwise global conformational changes were introduced by such mutations. Conformational features of bR with emphasis on surface area, as defined by surface complex, were discussed together with its biological significance, on the basis of the conformation-dependent displacement of the 13C chemical shifts. Further, slow local membrane dynamics with frequencies of 105 or 104 Hz, which is important for biological functions, was analyzed as viewed from specifically suppressed 13C NMR signals of certain regions caused by interference of fluctuation frequency with frequency of proton-decoupling or magic angle spinning. This approach has been further extended to reveal the conformation and dynamics of a variety of 13C-labeled membrane proteins that are overexpressed from Escherichia coli and subsequently not always involved in a 2D crystalline lattice by 13C NMR: they include phototaxis receptor protein (pharaonis phoborhodopsin), its transducer (pHtrII), and membrane enzyme (diacylglycerol kinase). In such cases, it was found that several 13C NMR signals could be suppressed from amino-acid residues located at the flexible portions such as loops and transmembrane α-helices near to the membrane surface, as a result of interference of dominant frequency for conformational fluctuations of the order of 104-105 Hz. This means that protein–protein contact in the 2D crystalline lattice significantly regulates the protein dynamics. It is therefore important to search for the most appropriate experimental condition to be able to observe the full 13C NMR signals of the membrane proteins under consideration, including the choice of the most appropriate 13C-labeled amino acids, temperature, ionic state, etc.