Participation of the surface structure of Pharaonis phoborhodopsin, ppR and its A149S and A149V mutants, consisting of the C-terminal α-helix and E-F loop, in the complex-formation with the cognate transducer pHtrII, as revealed by site-directed ¹³C solid-state NMR

We have recorded ¹³C solid state NMR spectra of [3- ¹³C]Ala-labeled pharaonis phoborhodopsin (ppR) and its mutants, A149S and A149V, complexed with the cognate transducer pharaonis halobacterial transducer II protein (pHtrII) (1-159), to gain insight into a possible role of their cytoplasmic surface structure including the C-terminal α-helix and E-F loop for stabilization of the 2:2 complex, by both cross-polarization magic angle spinning (CP-MAS) and dipolar decoupled (DD)-MAS NMR techniques. We found that ¹³C CP-MAS NMR spectra of [3-¹³C]Ala-ppR, A149S and A149V complexed with the transducer pHtrII are very similar, reflecting their conformation and dynamics changes caused by mutual interactions through the transmembrane α-helical surfaces. In contrast, their DD-MAS NMR spectral features are quite different between [3-¹³C]Ala- A149S and A149V in the complexes with pHtrII: ¹³C DD-MAS NMR spectrum of [3- ¹³C]Ala-A149S complex is rather similar to that of the uncomplexed form, while the corresponding spectral feature of A149V complex is similar to that of ppR complex in the C-terminal tip region. This is because more flexible surface structure detected by the DD-MAS NMR spectra are more directly influenced by the dynamics changes than the CP-MAS NMR. It turned out, therefore, that an altered surface structure of A149S resulted in destabilized complex as viewed from the ¹³C NMR spectrum of the surface areas, probably because of modified conformation at the corner of the helix E in addition to the change of hydropathy. It is, therefore, concluded that the surface structure of ppR including the C-terminal α-helix and the E-F loops is directly involved in the stabilization of the complex through conformational stability of the helix E.