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

Tolerance to freezing stress in cyanobacteria, Nostoc commune and some cyanobacteria with various tolerances to drying stress

Tolerance to and effects of the freezing stress in a desiccation-tolerant, terrestrial cyanobacterium, Nostoc commune, in cultivated strains of N. commune, and in desiccation-sensitive species, Synechocystis sp. PCC6803 and Fischerella muscicola, were studied by measuring their photosynthetic activities and fluorescence emission spectra. The results showed that a strain or species with higher desiccation tolerance was more tolerant to freezing stress than one with lower desiccation tolerance, which is consistent with the idea that tolerance to freezing stress is related to resistance to drying stress. Under freezing conditions, light energy absorbed by photosystem (PS) II complexes was dissipated to heat energy in N. commune, which may protect the cells from photoinactivation. N. commune encountered cellular dehydration due to ice formation outside the cell under freezing conditions. But NMR data showed that relatively high amounts of water still remained in a liquid state inside the cells at -36_C when N. commune colonies were fully wetted before freezing. High PSI activities measured by P700 photooxidation also support the result that non-freezing water remains within the cells. Besides, 5% methanol enhanced the resistance to freezing stress in the sensitive species. This effect seems to be related to maintenance of the PSI activity and pigment-protein complexes in their functional forms by methanol

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

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 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 (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 α-helices of the complexed [3-(13)C]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 (13)C CP-MAS spectra of [1-(13)C]Val-labeled complexed pHtrII showed that the (13)C 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 (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

Glutamic Acid Residues of Bacteriorhodopsin at the Extracellular Surface as Determinants for Conformation and Dynamics as Revealed by Site-Directed Solid-State (13)C NMR

We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacteriorhodopsin (bR) and a variety of its mutants, E9Q, E74Q, E194Q/E204Q (2Glu), E9Q/E194Q/E204Q (3Glu), and E9Q/E74Q/E194Q/E204Q (4Glu), to clarify contributions of the extracellular (EC) Glu residues to the conformation and dynamics of bR. Replacement of Glu-9 or Glu-74 and Glu-194/204 at the EC surface by glutamine(s) induced significant conformational changes in the cytoplasmic (CP) surface structure. These changes occurred in the C-terminal α-helix and loops, and also those of the EC surface, as viewed from (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled proteins. Additional conformational changes in the transmembrane α-helices were induced as modified retinal-protein interactions for multiple mutants involving the E194Q/E204Q pair. Significant dynamic changes were induced for the triple or quadruple mutants, as shown by broadened (13)C NMR peaks of [1-(13)C]Val-labeled proteins. These changes were due to acquired global fluctuation motions of the order of 10(−4)–10(−5) s as a result of disorganized trimeric form. In such mutants (13)C NMR signals from Val residues of [1-(13)C]Val-labeled triple and quadruple mutants near the CP and EC surfaces (including 8.7-Å depth from the surface) were substantially suppressed, as shown by comparative (13)C NMR studies with and without 40 μM Mn(2+) ion. We conclude that these Glu residues at the EC surface play an important role in maintaining the native secondary structure of bR in the purple membrane

Pressure induced isomerization of retinal on bacteriorhodopsin as disclosed by Fast Magic Angle Spinning NMR

Bacteriorhodopsin (bR), a retinal protein in purple membrane of H.salinarum, shows functions as a light-driven proton pump. We have detected pressure induced isomerization of retinal in bR by 15N cross polarization-magic angle spinning (CP-MAS) NMR spectra of [ε-15N]Lys-labeled bR. In the 15N NMR spectra, both all-trans and 13-cis retinal configurations have been observed at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 80 atm, the 15N NMR signals of Schiff Base of retinal were broadened. The signal intensity of 13-cis retinal at 155.0 ppm was increased when the MAS frequency was decreased from 12 kHz to 4 kHz. These results showed that the equilibrium constant of [13-cis –bR] /[all-trans-bR] increased by the pressure of 80 atm. It was also revealed that the changes induced by the pressure were quite local. Therefore, microscopically, hydrogen-bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated 13-cis isomerization of retinal in the membrane protein bR

Surface and dynamic structures of bacteriorhodopsin in a 2D crystal, a distorted or disrupted lattice, as revealed by site-directed solid-state 13C NMR

The 3D structure of bacteriorhodopsin (bR) obtained by x-ray diffraction or cryo-electron microscope studies is not always sufficient for a picture at ambient temperature where dynamic behavior is exhibited. For this reason, a site-directed solid-state 13C NMR study of fully hydrated bR from purple membrane (PM), or a distorted or disrupted lattice, is very valuable in order to gain insight into the dynamic picture. This includes the surface structure, at the physiologically important ambient temperature. Almost all of the 13C NMR signals are available from [3-13C]Ala or [1-13C]Val-labeled bR from PM, although the 13C NMR signals from the surface areas, including loops and transmembrane α-helices near the surface (8.7Å depth), are suppressed for preparations labeled with [1-13C]Gly, Ala, Leu, Phe, Tyr, etc. due to a failure of the attempted peak-narrowing by making use of the interfered frequency of the frequency of fluctuation motions with the frequency of magic angle spinning. In particular, the C-terminal residues, 226-235, are present as the C-terminal α-helix which is held together with the nearby loops to form a surface complex, although the remaining C-terminal residues undergo isotropic motion even in a 2D crystalline lattice (purple membrane) under physiological conditions. Surprisingly, the 13C NMR signals could be further suppressed even from [3-13C]Ala- or [1-13C]Val-bR, due to the acquired fluctuation motions with correlation times in the order of 10-4 to 10-5 s, when the 2D lattice structure is instantaneously distorted or completely disrupted, either in photo- intermediate, removed retinal or when embedded in the lipid bilayers