Izuru Kawamura

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

Site-Specific Solid-State NMR Detection of Hydrogen-Deuterium Exchange Reveals Conformational Changes in a 7-Helical Transmembrane Protein

Solid-state NMR spectroscopy is an efficient tool for following conformational dynamics of membrane proteins at atomic resolution. We used this technique for the site-specific detection of light-induced hydrogen-deuterium exchange in the lipid-embedded heptahelical transmembrane photosensor Anabaena sensory rhodopsin to pinpoint the location of its conformational changes upon activation. We show that the light-induced conformational changes result in a dramatic, but localized, increase in the exchange in the transmembrane regions. Most notably, the cytoplasmic half of helix G and the cytoplasmic ends of helices B and C exchange more extensively, probably as a result of their relative displacement in the activated state, allowing water to penetrate into the core of the protein. These light-induced rearrangements must provide the structural basis for the photosensory function of Anabaena sensory rhodopsin.

Structure and Orientation of Bovine Lactoferrampin in the Mimetic Bacterial Membrane as Revealed by Solid-State NMR and Molecular Dynamics Simulation

Bovine lactoferrampin (LFampinB) is a newly discovered antimicrobial peptide found in the N1-domain of bovine lactoferrin (268-284), and consists of 17 amino-acid residues. It is important to determine the orientation and structure of LFampinB in bacterial membranes to reveal the antimicrobial mechanism. We therefore performed (13)C and (31)P NMR, (13)C-(31)P rotational echo double resonance (REDOR), potassium ion-selective electrode, and quartz-crystal microbalance measurements for LFampinB with mimetic bacterial membrane and molecular-dynamics simulation in acidic membrane. (31)P NMR results indicated that LFampinB caused a defect in mimetic bacterial membranes. Ion-selective electrode measurements showed that ion leakage occurred for the mimetic bacterial membrane containing cardiolipin. Quartz-crystal microbalance measurements revealed that LFampinB had greater affinity to acidic phospholipids than that to neutral phospholipids. (13)C DD-MAS and static NMR spectra showed that LFampinB formed an α-helix in the N-terminus region and tilted 45° to the bilayer normal. REDOR dephasing patterns between carbonyl carbon nucleus in LFampinB and phosphorus nuclei in lipid phosphate groups were measured by (13)C-(31)P REDOR and the results revealed that LFampinB is located in the interfacial region of the membrane. Molecular-dynamics simulation showed the tilt angle to be 42° and the rotation angle to be 92.5° for Leu(3), which are in excellent agreement with the experimental values.

Structure and orientation of antibiotic peptide alamethicin in phospholipid bilayers as revealed by chemical shift oscillation analysis of solid state nuclear magnetic resonance and molecular dynamics simulation

The structure, topology and orientation of membrane-bound antibiotic alamethicin were studied using solid state nuclear magnetic resonance (NMR) spectroscopy. (13)C chemical shift interaction was observed in [1-(13)C]-labeled alamethicin. The isotropic chemical shift values indicated that alamethicin forms a helical structure in the entire region. The chemical shift anisotropy of the carbonyl carbon of isotopically labeled alamethicin was also analyzed with the assumption that alamethicin molecules rotate rapidly about the bilayer normal of the phospholipid bilayers. It is considered that the adjacent peptide planes form an angle of 100° or 120° when it forms α-helix or 310-helix, respectively. These properties lead to an oscillation of the chemical shift anisotropy with respect to the phase angle of the peptide plane. Anisotropic data were acquired for the 4 and 7 sites of the N- and C-termini, respectively. The results indicated that the helical axes for the N- and C-termini were tilted 17° and 32° to the bilayer normal, respectively. The chemical shift oscillation curves indicate that the N- and C-termini form the α-helix and 310-helix, respectively. The C-terminal 310-helix of alamethicin in the bilayer was experimentally observed and the unique bending structure of alamethicin was further confirmed by measuring the internuclear distances of [1-(13)C] and [(15)N] doubly-labeled alamethicin. Molecular dynamics simulation of alamethicin embedded into dimyristoyl phophatidylcholine (DMPC) bilayers indicates that the helical axes for α-helical N- and 310-helical C-termini are tilted 12° and 32° to the bilayer normal, respectively, which is in good agreement with the solid state NMR results.

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

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light‐driven proton pump. We have detected pressure‐induced isomerization of retinal in bR by analyzing 15N cross polarization‐magic angle spinning (CP‐MAS) NMR spectra of [ζ‐15N]Lys‐labeled bR. In the 15N‐NMR spectra, both all‐trans and 13‐cis retinal configurations have been observed in the Lys Nζ in protonated Schiff base 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 63 bar, the 15N‐NMR signals of [ζ‐15N]Lys in Schiff base of retinal were broadened. On the other hand, other [ζ‐15N]Lys did not show broadening. Subsequently, the increased signal intensity of [ζ‐15N]Lys in Schiff base of 13‐cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all‐trans‐bR]/[13‐cis‐bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. 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 isomerization of retinal from all‐trans to 13‐cis state in the membrane protein bR.

Dynamics Change of Phoborhodopsin and Transducer by Activation: Study Using D75N Mutant of the Receptor by Site-directed Solid-state 13C NMR†

Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative phototaxis receptor of Natronomonas pharaonis, and forms a complex, which transmits the photosignal into cytoplasm, with its cognate transducer (pHtrII). We examined a possible local dynamics change of ppR and its D75N mutant complexed with pHtrII, using solid‐state 13C NMR of [3‐13C]Ala‐ and [1‐13C]Val‐labeled preparations. We distinguished Ala Cβ13C signals of relatively static stem (Ala221) in the C‐terminus of the receptors from those of flexible tip (Ala228, 234, 236 and 238), utilizing a mutant with truncated C‐terminus. The local fluctuation frequency at the C‐terminal tip was appreciably decreased when ppR was bound to pHtrII, while it was increased when D75N, that mimics the signaling state because of disrupted salt bridge between C and G helices prerequisite for the signal transfer, was bound to pHtrII. This signal change may be considered with the larger dissociation constant of the complex between pHtrII and M‐state of ppR. At the same time, it turned out that fluctuation frequency of cytoplasmic portion of pHtrII is lowered when ppR is replaced by D75N in the complex with pHtrII. This means that the C‐terminal tip partly participates in binding with the linker region of pHtrII in the dark, but this portion might be released at the signaling state leading to mutual association of the two transducers in the cytoplasmic regions within the ppR/pHtrII complex.

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.

A Guide to Design Functional Molecular Liquids with Tailorable Properties using Pyrene-Fluorescence as a Probe

Solvent-free, nonvolatile, room-temperature alkylated-π functional molecular liquids (FMLs) are rapidly emerging as a new generation of fluid matter. However, precision design to tune their physicochemical properties remains a serious challenge because the properties are governed by subtle π-π interactions among functional π-units, which are very hard to control and characterize. Herein, we address the issue by probing π-π interactions with highly sensitive pyrene-fluorescence. A series of alkylated pyrene FMLs were synthesized. The photophysical properties were artfully engineered with rational modulation of the number, length, and substituent motif of alkyl chains attached to the pyrene unit. The different emission from the excimer to uncommon intermediate to the monomer scaled the pyrene-pyrene interactions in a clear trend, from stronger to weaker to negligible. Synchronously, the physical nature of these FMLs was regulated from inhomogeneous to isotropic. The inhomogeneity, unexplored before, was thoroughly investigated by ultrafast time-resolved spectroscopy techniques. The result provides a clearer image of liquid matter. Our methodology demonstrates a potential to unambiguously determine local molecular organizations of amorphous materials, which cannot be achieved by conventional structural analysis. Therefore this study provides a guide to design alkylated-π FMLs with tailorable physicochemical properties.

Color-Discriminating Retinal Configurations of Sensory Rhodopsin I by Photo-Irradiation Solid-State NMR Spectroscopy†

SRI (sensory rhodopsin I) can discriminate multiple colors for the attractant and repellent phototaxis. Studies aimed at revealing the color-dependent mechanism show that SRI is a challenging system not only in photobiology but also in photochemistry. During the photoreaction of SRI, an M-intermediate (attractant) transforms into a P-intermediate (repellent) by absorbing blue light. Consequently, SRI then cycles back to the G-state. The photoreactions were monitored with the (13)C NMR signals of [20-(13)C]retnal-SrSRI using in situ photo-irradiation solid-state NMR spectroscopy. The M-intermediate was trapped at -40 °C by illumination at 520 nm. It was transformed into the P-intermediate by subsequent illumination at 365 nm. These results reveal that the G-state could be directly transformed to the P-intermediate by illumination at 365 nm. Thus, the stationary trapped M- and P-intermediates are responsible for positive and negative phototaxis, respectively.

Drastic sensitivity enhancement in 29Si MAS NMR of zeolites and mesoporous silica materials by paramagnetic doping of Cu2

The paramagnetic doping of Cu(2+) in both mesoporous silica materials and microporous silicate crystals (zeolites) can be used effectively to enhance the signal intensity of (29)Si solid state magic angle spinning NMR, as a result of shortening of the spin-lattice relaxation time, T1, by the paramagnetic effect, because of the Cu(2+) electronic relaxation time in the range of 10(-8) s. This leads to drastically reduced data-collection times, typically 80-fold shorter than that in mesoporous silica. We found that the estimated range of the paramagnetic effect of Cu(2+) doping in porous silicates was at least 1 nm.