Structural Analysis of Transmembrane Halobacterial Tranceducer pHtrII by Multi-Dimensional High-Resolution Solid-State NMR

Abstract

°Transmembrane protein pHtrII is a transducer which binds to phoborhodopsin. The light excitation of phoborhodopsin is transmitted into the cytoplasm through pHtrII to promote negative photoaxis. We studied uniformly 13 C, 15 N labeled 159residues pHtrII by high-resolution solid-state NMR. The pHtrII (1-159) was econstructed into the deuterated DMPC membranes. High-resolution solid-state 2D NMR was measured under magic-angle spinning at the 1 H resonance frequencies of 500, 600 and 700 MHz . At first, we have assigned 13 C signals of pHtrII (1-159) by using 13 C- 13 C spin diffusion under DARR at -40 o C.Intraresidue correlations across 1-2 bonds were observed at short mixing time. At a long mixing time of 200 ms, intra-residue C α correlations across 3-bonds were obtained. Most amino acids except Tyr and Pro were assigned. The DARR spectra were observed for immobile region of pHtrII. All 13 C signals could not be observed in these dipolar-coupling-based cross-polarization methods because of large-amplitude molecular motions at room temperature. We performed Jcoupling-based HC-INEPT and HCC-TOCSY experiments in order to obtain signals of mobile protein segments. Mobile protein segments make up about 70% of pHtrII (1-159). Many signals in mobile regions were observed in HC and HCC 2D spectra. 13 C and 1 H signals were assigned to amino acid groups were successful in mobile regions. The C α and CO chemical shifts indicate that pHtrII (1-159) mainly forms α -helix structure in lipid bilayer environment.