Conformation and dynamics of the [3-(13)C]Ala, [1-(13)C]Val-labeled truncated pharaonis transducer, pHtrII(1-159), as revealed by site-directed (13)C solid-state NMR: changes due to association with phoborhodopsin (sensory rhodopsin II).
We have recorded (13)C NMR spectra of the [3-(13)C]Ala, [1-(13)C]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 (13)C NMR signals of [3-(13)C]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 alpha-helical peaks (alpha(II)-helix) and is identified with cytoplasmic alpha-helices protruding from the bilayers; the other was the high-field alpha-helical peak (alpha(I)-helix) and is identified with the transmembrane alpha-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 alpha-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 (13)C NMR spectra of [3-(13)C]Ala-labeled pHtrII. Consistent with this view, the (13)C DD-MAS NMR signals of the cytoplasmic alpha-helices of the complexed [3-(13)C]Ala-pHtrII in the dimyristoylphosphatidylcholine (DMPC) bilayer were partially suppressed at 0 degrees C due to a decreased fluctuation frequency at the low temperature. In contrast, examination of the (13)C CP-MAS spectra of [1-(13)C]Val-labeled complexed pHtrII showed that the (13)C NMR signals of the transmembrane alpha-helix were substantially suppressed. These spectral changes are again interpreted in terms of the increased fluctuation frequency of the transmembrane alpha-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 (13)C 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.
Yamaguchi
Satoru
S
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kamigori, Hyogo, Japan.
Shimono
Kazumi
K
Sudo
Yuki
Y
Tuzi
Satoru
S
Naito
Akira
A
Kamo
Naoki
N
Saitô
Hazime
H
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D001105:Archaea; D019843:Archaeal Proteins / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry; D002338:Carotenoids / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D025602:Halorhodopsins; D008051:Lipid Bilayers / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D025603:Sensory Rhodopsins; D015398:Signal Transduction; D013696:Temperature; D014633:Valine / Q000737:chemistry*
D019843:Archaeal Proteins; D025602:Halorhodopsins; D008051:Lipid Bilayers; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D002244:Carbon; D014633:Valine; D000409:Alanine; D004134:Dimyristoylphosphatidylcholine