Conformation and Dynamics of the [3- 13C]Ala, [1- 13C]Val-Labeled Truncated pharaonis Transducer, pHtrII(1–159), as Revealed by Site-Directed 13C Solid-State NMR: Changes Due to Association with Phoborhodopsin (Sensory Rhodopsin II)

Abstract

We have recorded 13C NMR spectra of the [3- 13C]Ala, [1- 13C]Val-labeled pharaonis transducer pHtrII(1–159) in the presence and absence of phoborhodopsin ( ppR or sensory rhodopsin II) in egg phosphatidylcholine or dimyristoylphosphatidylcholine bilayers by means of site-directed (amino acid specific) solid-state NMR. Two kinds of 13C NMR signals of [3- 13C]Ala- pHtrII complexed with ppR were clearly seen with dipolar decoupled magic angle spinning (DD-MAS) NMR. One of these resonances was at the peak position of the low-field α-helical peaks ( α II-helix) and is identified with cytoplasmic α-helices protruding from the bilayers; the other was the high-field α-helical peak ( α I-helix) and is identified with the transmembrane α-helices. The first peaks, however, were almost completely suppressed by cross-polarization magic angle spinning (CP-MAS) regardless of the presence or absence of ppR or by DD-MAS NMR in the absence of ppR. This is caused by an increased fluctuation frequency of the cytoplasmic α-helix from 10 5 Hz in the uncomplexed states to >10 6 Hz in the complexed states, leading to the appearance of peaks that were suppressed because of the interference of the fluctuation frequency with the frequency of proton decoupling (10 5 Hz), as viewed from the 13C NMR spectra of [3- 13C]Ala-labeled pHtrII. Consistent with this view, the 13C DD-MAS NMR signals of the cytoplasmic α-helices of the complexed [3- 13C]Ala- pHtrII in the dimyristoylphosphatidylcholine (DMPC) bilayer were partially suppressed at 0°C due to a decreased fluctuation frequency at the low temperature. In contrast, examination of the 13C CP-MAS spectra of [1- 13C]Val-labeled complexed pHtrII showed that the 13C NMR signals of the transmembrane α-helix were substantially suppressed. These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane α-helices from 10 3 Hz of the uncomplexed states to 10 4 Hz of the complexed states. These findings substantiate the view that the transducers alone are in an aggregated or clustered state but the ppR- pHtrII complex is not aggregated. We show that 13C NMR is a very useful tool for achieving a better understanding of membrane proteins which will serve to clarify the molecular mechanism of signal transduction in this system.