Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

AbstractWe have recorded 13C nuclear magnetic resonance (NMR) spectra of [3-13C]Ala, [1-13C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven 13C NMR signals from transmembrane α-helices were resolved for [3-13C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, 13C NMR signals from the loops were visible from [1-13C]Val-ppR but their peak positions of the transmembrane α-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 105 Hz in view of the suppressed peaks from [3-13C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1–159). In particular, the C-terminal α-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude

Dynorphin induced magnetic ordering in lipid bilayers as studied by 31P NMR spectroscopy

AbstractLipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1–17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (Tm=24°C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1–13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1–17) near at Tm but not for the system containing A(1–13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at Tm. These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1–17). It turned out that the bilayers are spontaneously aligned to the magnetic field above Tm in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1–13). It was proved that DMPC bilayer in the presence of dynorphin A(1–17) forms vesicles above Tm, because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape

Morphological Behavior of Lipid Bilayers Induced by Melittin near the Phase Transition Temperature

Morphological changes of DMPC, DLPC, and DPPC bilayers containing melittin (lecithin/melittin molar ratio of 10:1) around the gel-to-liquid crystalline phase transition temperatures (Tc) were examined by a variety of biophysical methods. First, giant vesicles with the diameters of ∼20 μm were observed by optical microscopy for melittin-DMPC bilayers at 27.9°C. When the temperature was lowered to 24.9°C (Tc = 23°C for the neat DMPC bilayers), the surface of vesicles became blurred and dynamic pore formation was visible in the microscopic picture taken at different exposure times. Phase separation and association of melittin molecules in the bilayers were further detected by fluorescent microscopy and mass spectrometry, respectively. These vesicles disappeared completely at 22.9°C. It was thus found that the melittin-lecithin bilayers reversibly undergo their fusion and disruption near the respective Tcs. The fluctuation of lipids is, therefore, responsible for the membrane fusion above the Tc, and the association of melittin molecules causes membrane fragmentation below the Tc. Subsequent magnetic alignments were observed by solid-state (31)P NMR spectra for the melittin-lecithin vesicles at a temperature above the respective Tcs. On the other hand, additional large amplitude motion induced by melittin at a temperature near the Tc breaks down the magnetic alignment