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Alzheimer’s disease (AD) is associated with progressive accumulation of amyloid-β (Aβ) cross-β fibrils in the brain. Aβ species tightly associated with GM1 ganglioside, a glycosphingolipid abundant in neuronal membranes, promote amyloid fibril formation; therefore, they could be attractive clinical targets. However, the active conformational state of Aβ in GM1-containing lipid membranes is still unknown. The present solid-state nuclear magnetic resonance study revealed a nonfibrillar Aβ assemblage characterized by a double-layered antiparallel β-structure specifically formed on GM1 ganglioside clusters. Our data show that this unique assemblage was not transformed into fibrils on GM1-containing membranes, but could promote conversion of monomeric Aβ into fibrils, suggesting that a solvent-exposed hydrophobic layer provides a catalytic surface evoking Aβ fibril formation. Our findings will offer structural clues for designing drugs targeting catalytically active Aβ conformational species for the development of anti-AD therapeutics.

Mallory reaction of 1,2-bis(4-hydroxyphenyl)-3,3,4,4,5,5-hexafluorocyclopentene under light irradiation (λ = 356 nm) in the presence of iodine as an oxidant proceeded to give fluorine-containing phenanthrene bearing hydroxyl groups, 6,9-bis(4-hydroxyphenyl)-1,1,2,2,3,3-hexafluoro-2,3-dihydro-1H-cyclopenta[l]phenanthrene. Polycondensation of the prepared fluorine-containing phenanthrene with various acid dichlorides afforded the phenanthrene-type polyesters. For comparison, diarylethene-type polyesters were also prepared analogously. Diarylethene-type polyesters consisting m-phenylene unit showed good solubility in typical organic solvents such as CHCl3. Tetramethylene-linked diarylethene-type polyesters with Mn = 4100 (Mn = number average molecular weight) was isolated as oil. Other diarylethene-type and all phenanthrene-type polyesters were insoluble in organic solvents. The phenanthrene-type polyesters had high thermal stability revealed by TGA analysis.

A one‐handed 310‐helical hexapeptide is efficiently encapsulated within the helical cavity of st‐PMMA when a fullerene (C60) derivative is introduced at the C‐terminal end of the peptide. The encapsulation is accompanied by induction of a preferred‐handed helical conformation in the st‐PMMA backbone with the same‐handedness as that of the hexapeptide to form a crystalline st‐PMMA/peptide‐C60 inclusion complex with a unique optically active helix‐in‐helix structure. Although the st‐PMMA is unable to encapsulate the 310‐helical peptide without the terminal C60 unit, the helical hollow space of the st‐PMMA is almost filled by the C60‐bound peptides. This result suggests that the C60 moiety can serve as a versatile molecular carrier of specific molecules and polymers in the helical cavity of the st‐PMMA for the formation of an inclusion complex, thus producing unique supramolecular soft materials that cannot be prepared by other methods.

Poly-l-alanine (PLA) sequences are a key element in the structure of the crystalline domains of spider dragline silks, wild silkworm silks, antifreeze proteins, and amyloids. To date, no atomic-level structures of antiparallel (AP)-PLA longer than Ala4 have been reported using the single-crystal X-ray diffraction analysis. In this work, dipolar-assisted rotational resonance solid-state NMR spectra were observed to determine the effective internuclear distances of 13C uniformly labeled alanine tetramer with antiparallel (AP) β-sheet structure whose atomic coordinates are determined from the X-ray crystallographic analysis. Initial build-up rates, Rj,k, were obtained from the build-up curves of the cross peaks by considering the internuclear distances arising in the master equation. Subsequently, experimentally obtained effective internuclear distances, reffj,k(obs), were compared with the calculated reffj,k(calc) values obtained from the X-ray crystallographic data. Fairly good correlation between reffj,k(...

A palladium-catalyzed alternating copolymerization of [2.2.1]oxabicyclic alkenes 1 with carbon monoxide afforded isomer mixtures of polyketones 2ktn and polyketals 2ktl. Subsequent acid-induced dehydration of polymer 2 furnished novel aromatic polymers consisting of polyketones 3ktn and polyketals 3ktl units. This formal aryne/carbon monoxide copolymerization thus generated the first example of poly(aryne-alt-carbon monoxide)s, in which o-arylene and carbonyl units are incorporated in an alternating fashion.

Regulation of electron transfer on organic substances by external stimuli is a fundamental issue in science and technology, which affects organic materials, chemical synthesis, and biological metabolism. Nevertheless, acid/base-responsive organic materials that exhibit reversible electron transfer have not been well studied and developed, owing to the difficulty in inventing a mechanism to associate acid/base stimuli and electron transfer. We discovered a new phenomenon in which N–N linked bicarbazole (BC) and tetramethylbiacridine (TBA) derivatives undergo electron transfer disproportionation by acid stimulus, forming their stable radical cations and reduced species. The reaction occurs through a biradical intermediate generated by the acid-triggered N–N bond cleavage reaction of BC or TBA, which acts as a two electron acceptor to undergo electron transfer reactions with two equivalents of BC or TBA. In addition, in the case of TBA the disproportionation reaction is highly reversible through neutralization with NEt3, which recovers TBA through back electron transfer and N–N bond formation reactions. This highly reversible electron transfer reaction is possible due to the association between the acid stimulus and electron transfer via the acid-regulated N–N bond cleavage/formation reactions which provide an efficient switching mechanism, the ability of the organic molecules to act as multi-electron donors and acceptors, the extraordinary stability of the radical species, the highly selective reactivity, and the balance of the redox potentials. This discovery provides new design concepts for acid/base-regulated organic electron transfer systems, chemical reagents, or organic materials.

We have previously suggested that crystalline Bombyx mori silk in silk II form (the silk structure after spinning) is not a simple antiparallel β-sheet but is intrinsically heterogeneous. Using the peptide (AG)15, we have obtained the first fully assigned high resolution solid state 1H NMR spectrum. Distinct heterogeneity was observed, in both 1H and 13C CP/MAS signals. Based on these results, a new model is proposed that contains two different packing arrangements of antiparallel β-sheets. The structures were energetically minimized by CASTEP calculation and used to calculate the solid state 1H, 13C, and 15N NMR chemical shifts using the GIPAW method. This new model was supported by good agreement between the calculated and observed 1H, 13C, and 15N chemical shifts and relative 1H–1H proximities obtained from 2D 1H DQMAS experiments. We conclude that the intermolecular packing of B. mori silk fibroin has been finally resolved.

The accurate 1H positions of alanine-glycine alternating copolypeptide, (AG)15 with Silk I structure were determined. For the purpose, the geometry optimization was performed starting with the atomic coordinates of the hetero atoms reported previously (Macromolecules 2005, 38, 7397−7403) and applied only for protons under periodic boundary conditions. The agreement between the calculated and observed chemical shifts of all 1H,13C and 15N nuclei was excellent, indicating strongly that the determination of all the atomic-coordinate including 1H nuclei was performed with high accuracy. Here the 1H chemical shift was obtained by using both 1 mm microcoil MAS NMR probe-head for mass-limited solid-state samples developed by us and ultrahigh field NMR at 920 MHz. The DQ correlations in the 1H DQMAS NMR spectra were also used to confirm the intra- and intermolecular structures obtained here. The characteristic structure of Silk I which can be easily converted to Silk II by external forces was discussed together w...

The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP(3)-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP(3) and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP(3)-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.

Covalent organic frameworks are a class of crystalline porous polymers that integrate molecular building blocks into periodic structures and are usually synthesized using two-component [1+1] condensation systems comprised of one knot and one linker. Here we report a general strategy based on multiple-component [1+2] and [1+3] condensation systems that enable the use of one knot and two or three linker units for the synthesis of hexagonal and tetragonal multiple-component covalent organic frameworks. Unlike two-component systems, multiple-component covalent organic frameworks feature asymmetric tiling of organic units into anisotropic skeletons and unusually shaped pores. This strategy not only expands the structural complexity of skeletons and pores but also greatly enhances their structural diversity. This synthetic platform is also widely applicable to multiple-component electron donor–acceptor systems, which lead to electronic properties that are not simply linear summations of those of the conventional [1+1] counterparts., Covalent organic frameworks are crystalline porous polymers integrating molecular building blocks into periodic structures. Here, the authors report a general multiple-component condensation strategy that enables the use of one knot and two or three linkers to synthesize complex, anisotropic frameworks.

Various neurodegenerative disorders are ascribed to pathogenic molecular processes involving conformational transitions of amyloidogenic proteins into toxic aggregates characterized by their β structures. Accumulating evidence indicates that neuronal cell membranes provide platforms for such conformational transitions of pathogenic proteins as best exemplified by amyloid β (Aβ). Therefore, membrane-bound Aβ species can be promising targets for the development of novel drugs for Alzheimer’s disease. In the present study, solid-state nuclear magnetic resonance spectroscopy has elucidated the membrane-induced conformation of Aβ, in which the disordered N-terminal segment is followed by the stable C-terminal β strand. The data provides an insight into the molecular processes of the conformational transition of Aβ coupled with its assembly into parallel β structures.

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.

2H (I = 1) quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) NMR methods in the presence of a paramagnetic shift interaction are reported. Pulse sequences based on quadrupole echo that can compensate effect of the shift interaction were adapted to obtain undistorted QCPMG spectra. The methods were demonstrated by 2H NMR measurement for paramagnetic CoSiF6·6H2O using strong radio-frequency pulses and by computer simulation. Detection of molecular dynamics by the present QCPMG method was also examined.

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state (31)P and (13)C NMR spectroscopy. (31)P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (T(c)) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the T(c). (13)C NMR spectra of site-specifically (13)C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers.

We report solid-state 95Mo NMR of Mo V , Mo V,VI and Mo VI species in mixed-valence polyoxomolybdates (V, VI) with localized or delocalized d 1 electrons. Parameters about chemical shift and quadrupole interactions of 95Mo in diamagnetic crystals of [ Me 3 NH ] 6 [ H 2 Mo 12 V O 28 ( OH ) 12 ( Mo VI O 3 ) 4 ] · 2 H 2 O ( 1 ) with localized electrons and [ NMe 4 ] 2 [ NH 4 ] 8 [ ( Mo 6 VI Mo V O 23 ) 2 ] · 8 H 2 O ( 2 ) with delocalized ones were obtained by simulation of magic-angle-spinning (MAS) 95Mo NMR spectra. The isotropic and anisotropic chemical shifts of sites of Mo V in 1 and Mo V, VI in 2, respectively, exhibited absolute values quite larger than those of other Mo VI sites, which is examined by quantum chemical calculations.

The effects of geometric properties of membranes on the structure of the phospholipase C-delta1 (PLC-delta1) pleckstrin homology (PH) domain were investigated using solid state (13)C NMR spectroscopy. Conformations of the PLC-delta1 PH domain at the surfaces of multilamellar vesicles (MLV), small unilamellar vesicles (SUV), and micelles were examined to evaluate the effects of membrane curvature on the PH domain. An increase in curvature of the water-hydrophobic layer interface hinders membrane-penetration of the amphipathic alpha2-helix of the PH domain that assists the membrane-association of the PH domain dominated by the phosphatidylinositol 4,5-bisphosphate (PIP(2)) specific lipid binding site. The solid state (13)C NMR signal of Ala88 located at the alpha2-helix indicates that the conformation of the alpha2-helix at the micelle surface is similar to the solution conformation and significantly different from those at the MLV and SUV surfaces which were characterized by membrane-penetration and re-orientation. The signal of Ala112 which flanks the C-terminus of the beta5/beta6 loop that includes the alpha2-helix, showed downfield displacement with decrease in the interface curvature of the micelles, SUV and MLV. This reveals that the conformation of the C-terminus of the beta5/beta6 loop connecting the beta-sandwich core containing the PIP(2) binding site and the amphipathic alpha2-helix is sensitive to alterations of the curvature of lipid bilayer surface. It is likely that these alterations in the conformation of the PLC-delta1 PH domain contribute to the regulatory mechanisms of the intracellular localization of PLC-delta1 in a manner dependent upon the structure of the molecular complex containing PIP(2).

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 [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) 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 [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-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.

11 B and 23 Na solid-state nuclear magnetic resonance (NMR) spectra of ring-shaped paramagnetic crystals of H 15 [ V 7 IV V 5 V B 32 O 84 Na 4 ] · 13 H 2 O containing seven d 1 electrons from V IV were studied. Magic-angle-spinning (MAS) and multiple-quantum MAS NMR experiments were performed at moderate (9.4 T) and ultrahigh magnetic fields (21.6 T). The NMR parameters for quadrupole and isotropic chemical shift interactions were estimated by simulation of the NMR spectra and from relativistic density functional theory (DFT) calculations. Four Na ions incorporated into the framework were found to occupy four distinct sites with different populations. The DFT calculation showed that d 1 electrons with effectively one up-spin caused by strong antiferromagnetic interactions were delocalized over the 12 V ions.

We propose a novel approach to markedly reduce rf power for both 1H and observed nuclei during spin exchange for separated local field experiments. The rf power to satisfy the Hartmann–Hahn matching conditions during spin exchange for observed nuclei was arbitrarily reduced by alternating the directions of effective fields for 1H nuclei with unequal duration times and amplitudes. The proposed techniques were compared experimentally with those developed previously by the authors. The rf power for observed nuclei and average 1H were reduced by factors of 9 and 2, respectively, for 13C NMR signals of liquid crystalline 5CB.

Secondary structure and orientation of dynorphin bound to dimyristoylphosphatidylcholine (DMPC) bilayer were investigated by solid-state 13C NMR spectroscopy. For this purpose, 13C NMR spectra of the site-specifically 13C-labeled dynorphin were measured in the membrane-bound state under static, magic angle spinning (MAS), and slow MAS conditions. In the static experiment, magnetically oriented vesicle system (MOVS) induced by dynorphin was successfully used to investigate the orientation of dynorphin bound to the lipid bilayers. It was found that dynorphin adopts an α-helical structure in the N-terminus from Gly2 to Leu5 by analyses of the isotropic chemical shifts obtained from the MAS experiments. In contrast, it adopts disordered conformations from the center to the C-terminus and is located on the membrane surface. The static 13C NMR spectra indicated that MOVS-bound dynorphin was oriented to the magnetic field and rotated rapidly about the bilayer normal. Subsequently, we analyzed the 13C chemical shift tensors of carbonyl carbons in the peptide backbone by considering the rotational motion of the N-terminal α-helix. It was revealed that the N-terminal α-helix is inserted into the membrane with the tilt angle of 21° to the bilayer normal. This structure suggests a possibility that dynorphin interacts with the extracellular loop II of the κ-receptor through a helix–helix interaction.

A new method for two-dimensional separated local field solid-state NMR spectroscopy was developed. By applying a time averaging effect for the nutation frequency in the tilted rotating frame, which we proposed recently, a large reduction of the RF power for observed nuclei during the spin exchange period can be achieved without impairing the 1H homonuclear dipolar decoupling efficiency. The reduction of the RF nutation frequency and the RF field for observed nuclei during the spin exchange period were achieved experimentally as 1/3 and 1/9, respectively, for the 13C NMR signals of liquid-crystalline 5CB. Consequently, effective attenuation of sample heating was obtained.

Solid-state 31P- and 13C-NMR spectra were recorded in melittin-lecithin vesicles composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Highly ordered magnetic alignments were achieved with the membrane surface parallel to the magnetic field above the gel-to-liquid crystalline phase transition temperature (Tc). Using these magnetically oriented vesicle systems, dynamic structures of melittin bound to the vesicles were investigated by analyzing the 13C anisotropic and isotropic chemical shifts of selectively 13C-labeled carbonyl carbons of melittin under the static and magic-angle spinning conditions. These results indicate that melittin molecules adopt an alpha-helical structure and laterally diffuse to rotate rapidly around the membrane normal with tilt angles of the N-terminal helix being -33 degrees and -36 degrees and those of the C-terminal helix being 21 degrees and 25 degrees for DLPC and DPPC vesicles, respectively. The rotational-echo double-resonance method was used to measure the interatomic distance between [1-13C]Val8 and [15N]Leu13 to further identify the bending alpha-helical structure of melittin to possess the interhelical angles of 126 degrees and 119 degrees in DLPC and DPPC membranes, respectively. These analyses further lead to the conclusion that the alpha-helices of melittin molecules penetrate the hydrophobic cores of the bilayers incompletely as a pseudo-trans-membrane structure and induce fusion and disruption of vesicles.

We report a simple two-dimensional NMR method for obtaining 2H NMR pure-quadrupole spectra of paramagnetic solids. This method is based on a quadrupole-echo sequence inserted with 180° pulses, where the pulse spacings are incremented asymmetrically so that the 2H magnetization evolves only by the quadrupole interaction in the indirect dimension. It is shown that when the sequence is carried out with strong radio-frequency pulses, the spectrum projected to the indirect dimension can be simulated using the quadrupole-echo sequence without considering the effect of the paramagnetic shift. The method was also used to investigate molecular dynamics by measurement and simulation of the temperature dependence of the 2H NMR spectra.

Practical heteronuclear dipolar recoupling performances under magic angle spinning for SFAM and REDOR have been investigated under well-defined rf inhomogeneity environments with variation of resonance offsets for the irradiated nucleus. The heteronuclear dipolar recoupling efficiencies were quantitatively determined based on the experimentally obtained rf homogeneity. As a result, SFAM retains higher recoupling efficiency (>95%) at an 85% effective nutation frequency, and its recoupling efficiency is gradually reduced at lower effective nutation frequencies. On the other hand, although REDOR retains higher recoupling (>95%) efficiency at high (>92%) effective nutation frequency with an XY-8 compensation pulse sequence, the recoupling efficiency is dramatically decreased when the effective nutation frequency is below 90%. Over all, SFAM has significant advantages for insensitivity to carrier frequency offset and rf inhomogeneity.

Interatomic distances of isotopically doubly labeled N-Acetyl-Pro-Gly-Phe crystals, obtained from rotational echo double resonance (REDOR) experiments, were analyzed as a three-spin system in which observed nuclei are simultaneously coupled with isotopically labeled intra- and intermolecular heteronuclei. In particular, the intermolecular dipolar interaction was examined to obtain a clue as to the relative orientation of a given molecule to the surrounding neighbors. We have developed a systematic procedure to determine both the intra- and intermolecular interatomic distances and the angle between these two interatomic vectors. The former intramolecular distances were determined by fitting the infinitely diluted normalized REDOR differences ΔS/S0 values to the theoretical curve as a two-spin system. Then, ΔS/S0 values from undiluted doubly labeled samples were analyzed as three-spin systems. Subsequently, the interatomic distances and the angles, between the two isotopically labeled nuclei belonging to th...

We have determined the three-dimensional structure of [13C,15N]-labeled Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) dihydrate (crystallized from aqueous methanol) on the basis of six sets of accurately determined 13C···15N interatomic distances by rotational echo double resonance (REDOR) and some additional constraints from 13C chemical shifts. This compound has not yet been refined by X-ray diffraction. Six kinds of [13C,15N]-doubly-labeled samples, in which the doubly-labeled positions are four-bonds apart (four samples) and five-bonds apart (two samples), were chemically synthesized. These labeled peptides (100%) and an isotopically diluted one with unlabeled samples (60% or 30%) were crystallized from aqueous methanol solution. 13C or 15N chemical shifts were carefully evaluated prior to and after every REDOR experiment in order to check that the crystalline polymorphs under consideration were not modified either by loss of or by freezing of motion of solvent molecules in the crystals. Accurate and precise i...

We have explored an empirical approach to clarify the three-dimensional structure and dynamics of membrane proteins such as bacteriorhodopsin (bR) based on 13C NMR measurements, utilizing the concept of the conformation-dependent displacements of 13C chemical shifts as determined by cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) methods. This is possible because 13C chemical shifts of the amino acid residues under consideration are appreciably displaced, up to 8 ppm, depending upon particular conformations from several portions of membrane proteins such as the transmembrane α-helices, loops, N- or C-terminus, etc. as referred to the data accumulated to date of an appropriate model system. It is also possible to distinguish a region characterized by a variety of backbone motions with at least three different time scales from NMR data: rapid motions with correlation times shorter than 10−8 s, intermediate motions with correlation times of 10−4 to 10−5 s, and slow motions with a time scale of 10−2 s. In addition, we also explored a non-empirical approach to reveal the three-dimensional structure of a smaller molecular system such as biologically active peptides as a messenger molecule in signal transduction, based on accurately determined appropriate sets of interatomic distances as determined by rotational echo double resonance (REDOR). Examination of 13C or 15N chemical shifts before and after REDOR experiments proved to be an indispensable means to examine whether or not conformations of several kinds of 13C, 15N-doubly labeled samples at different positions are not changed all the time. Here, we summarize some illustrative examples to this end, selected from our recent studies on [3-13C]Ala-labeled bR and biologically active peptides such as enkephalins.

The interatomic distances in the crystalline specimen of 13C,15N doubly labeled peptides [1-13C]N-acetyl-Pro-[15N]Gly-Phe (I), N-acetyl-[1-13C]Pro-Gly-[15N]Phe (II), and [1-13C]N-acetyl-Pro-Gly-[15N]Phe (III) evaluated from rotational echo double resonance (REDOR) data were compared with those from X-ray diffraction studies and justify our novel approach. The minimization of B1 inhomogeneity was critical to obtain accurate distances, which were achieved by confinement of the samples in the central portion (50% of the total filling volume of the rotor). The effect of the finite length of the π pulse was found to be negligible as long as the pulse length is less than 10% of the rotor cycle. The 13C···15N distances obtained from 13C REDOR were thus 3.24 ± 0.05, 3.43 ± 0.05, and 4.07 ± 0.05 A for I, II, and III, respectively. The REDOR-derived conformation of this peptide was β-turn type I, consistent with our X-ray diffraction study (orthorhombic crystal). The maximum deviation of the distances determined by...

Alanine oligomers provide a key structure for silk fibers from spider and wild silkworms.We report on structural analysis of L-alanyl-L-alanyl-L-alanyl-L-alanine (Ala)4 with anti-parallel (AP) β-structures using X-ray and solid-state NMR. All of the Ala residues in the (Ala)4 are in equivalent positions, whereas for alanine trimer (Ala)3 there are two alternative locations in a unit cell as reported previously (Fawcett and Camerman, Acta Cryst., 1975, 31, 658-665). (Ala)4 with AP β-structure is more stable than AP-(Ala)3 due to formation of the stronger hydrogen bonds. The intermolecular structure of (Ala)4 is also different from polyalanine fiber structure, indicating that the interchain arrangement of AP β-structure changes with increasing alanine sequencelength. Furthermore the precise (1)H positions, which are usually inaccesible by X-ray diffraction method, are determined by high resolution (1)H solid state NMR combined with the chemical shift calculations by the gauge-including projector augmented wave method.

Protein activities are generally regulated by intramolecular allosteric interactions, by which spatially separated sites in a protein molecule communicate. Intramolecular allosteric interactions in the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain were investigated by solution NMR spectroscopy for selectively [α-15N]Lys-labeled proteins. The results of NMR analyses indicated that the binding of inositol 1,4,5-trisphosphate (IP3) to the protein induces local environmental changes at all lysine residues, including residues such as Lys-43 spatially separated from the specific IP3 binding site consisting of Lys-30, Lys-32, and Lys-57. IP3 binding also induces conformational stabilization of a characteristic short α-helix (α2) from residues 82 to 87. Mutational analyses indicated that an interaction network mainly consisting of the side chains of Lys-30, Lys-32, and Lys-43 exists in the ligand-free protein, and it was therefore predicted that binding of IP3 to the specific site modifies the interaction network, resulting in formation of a new interaction network, in which the side chains of Lys-57 and Phe-87 contribute to stable IP3 binding. These results provide evidence for intramolecular interactions in the PLC-δ1 PH domain, the function of which could be allosterically regulated by modifications at sites spatially separated from the ligand-binding site through the intramolecular interaction network.

Both 13 C- and 15 N-REDOR experiments were performed to determine the 13 C- 15 N interatomic distance in [ 1- 13 C]N-acetyl-Pro-[ 15 N]Gly-Phe crystal and to evaluate the inter- or intra-molecular dipolar contributions leading to its errors. The interatomic distance was determined to be 3.43 A after removing the intermolecular contribution to the echo dephasing arising from the intermolecular dipolar interaction with labeled nuclei of the neighboring molecule. This contribution was estimated by extrapolation to infinite dilution of the labeled sample in unlabeled peptide. The theoretical formalism was developed for the nucleus coupled with two heteronuclei. The conformation of this peptide turned out to be β-turn type II on the basis of the abovementioned interatomic distance.

We report on experimental results from the heavy ion CT (IonCT) system based on the measurement of residual range distribution; this technique was first investigated by Zygmanski et al. (2000) for proton beams. Experiments were done on the spatial and density resolution phantom by using 4He and 12C beams accelerated up to 230 MeV/u and 400 MeV/u by the Heavy Ion Medical Accelerator in Chiba (HIMAC), installed in the National Institute of Radiological Sciences (NIRS) in Japan, with a high performance intensified charged coupled device (ICCD) camera. We show that rods with a diameter of 1 mm can be successfully reproduced using the 12C beam with this technique. We also show that the MCS blurring effect in the reconstructed image can be reduced for the heavier ion beam. The reduction in the background neutron noise from the phantom and/or collimator, by using the data obtained from the improvement of the optical detector system in 2007, is also shown.

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 [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) 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 [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-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.

The heart of the H + conductance mechanism in the homotetrameric M2 H + channel from influenza A is a set of four histidine side chains. Here, we show that protonation of the third of these imidazoles coincides with acid activation of this transmembrane channel and that, at physiological pH, the channel is closed by two imidazole–imidazolium dimers, each sharing a low-barrier hydrogen bond. This unique construct succeeds in distributing a pair of charges over four rings and many atoms in a low dielectric environment to minimize charge repulsion. These dimers form with identical pK a s of 8.2 ± 0.2, suggesting cooperative H + binding and clearly illustrating high H + affinity for this channel. The protonation behavior of the histidine side chains has been characterized by using solid-state NMR spectroscopy on the M2 transmembrane domain in fully hydrated lipid bilayers where the tetrameric backbone structure is known. Furthermore, electrophysiological measurements of multichannel and single-channel experiments confirm that these protein constructs are functional.

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.

Determination of conformations and structures of opioid peptides in the membrane environments is an essential step to understand the action of the peptide to the specialized receptors. This information not only gains insight into the structure-function relationship of opioid peptide but also gives proper guidelines to design a new drug to have same neuroendocrine functions. This review provides the structural studies of three types of opioid peptide families such as enkephalin, beta-endorphin and dynorphin in the solid states and the membrane environments. The structures of enkephalins show that they take beta-bend, extended and double beta-bend structures in the crystals. Moreover, enkephalin molecules take a variety of structures in the crystals and are easily converted to the other structures with slightly different torsion angles. On the other hand, beta-bend structures are mostly seen in the membrane environments. Membrane bound structure of dynorphin shows that the N-terminus forms alpha-helical structure and is inserted into the membrane with the helical axis almost perpendicular to the membrane surface. It is discussed that the helical region of the extracellular loop II of the kappa-opioid receptor may interact with the helical region of dynorphin with a high affinity in the membrane environments. beta-endorphin takes alpha-helical structure at N-terminus and the central regions and the rest of regions take unordered structure when the bind to the membrane. Since the membrane bound structures of opioid peptides differ from those of the solution states, membrane association is an important process for exerting the affinity and the selectivity to the specific opioid receptors.

A fluorescent x‐ray source for medical imaging, such as K‐edge subtraction angiography and monochromatic x‐ray CT, has been developed. Using a 6.5 GeV accumulation ring in Tsukuba, fluorescent x rays, which range from about 30 to 70 keV are generated by irradiating several target materials. Measurements have been made of output intensities and energy spectra for different target angles and extraction angles. The intensities of fluorescent x rays at a 30 mA beam current are on the order of 1–3×106 photons/mm2/s at 30 cm from the local spot where the incident beam is collimated to 1 mm2. A phantom which contains three different contrast media (iodine, barium, gadolinium) was used for the K‐edge energy subtraction, and element selective CT images were obtained.

The relative electron density resolution was discussed by the Wiener spectrum in the heavy ion CT image. The two-dimensional (2D) Wiener spectrum in the CT image was obtained from the one-dimensional (1D) Wiener spectrum of the measured residual range distribution of the water phantom for a single projection angle, and the relative electron density resolution in the CT image was calculated from the 2D Wiener spectrum. To examine the usefulness of this method, the relative electron density resolution was also estimated by other two methods; the calculation using the Wiener spectrum of the reconstructed image of the water phantom, and the estimation by the reconstructed image of the electron density resolution phantom. The result of the first method was similar to those of the other two methods. Therefore, it is useful to estimate the relative electron density resolution by the 1D Wiener spectrum of the measured residual range distribution of the water phantom for a single projection angle.

An algorithm for heavy ion CT reconstruction from residual range measurement data is presented. The experimental procedure is described, in which an intensifying screen and a TV camera with a micro-channel-plate intensifier were used for the detection of energy deposition profile for the heavy ion /sup 12/C with an initial energy of 400 MeV/u emitted from a HIMAC. A series of projection images of a water phantom with PMMA pipes were experimentally taken for various values of range shifter thickness at each projection angle. From these images the distribution of residual range was obtained and used for reconstruction of the relative stopping power. The spatial resolution of the system was estimated as less than 2 mm.

An interhelical distance has been precisely measured by REDOR solid-state NMR spectroscopy in the transmembrane tetrameric bundle of M2-TMP, from the M2 proton channel of the influenza A viral coat. The high-resolution structure of the helical backbone has been determined using orientational restraints from uniformly aligned peptide preparations in hydrated dimyristoylphosphatidylcholine bilayers. Here, the distance between (15)N(pi) labeled His37 and (13)C(gamma) labeled Trp41 is determined to be less than 3.9 A. Such a short distance, in combination with the known tilt and rotational orientation of the individual helices, permits not only a determination of which specific side chain pairings give rise to the interaction, but also the side chain torsion angles and restraints for the tetrameric bundle can also be characterized. The resulting proton channel structure is validated in a variety of ways. Both histidine and tryptophan side chains are oriented in toward the pore where they can play a significant functional role. The channel appears to be closed by the proximity of the four indoles consistent with electrophysiology and mutagenesis studies of the intact protein at pH 7.0 and above. The pore maintains its integrity to the N terminal side of the membrane, and at the same time, a cavity is generated that appears adequate for binding amantadine. Finally, the observation of a 2 kHz coupling in the PISEMA spectrum of (15)N(pi)His37 validates the orientation of the His37 side chain based on the observed REDOR distance.