Microscopic vibrational circular dichroism on the forewings of a European hornet: heterogenous sequences of protein domains with different secondary structures.

We developed a microscopic scanning for vibrational circular dichroism (VCD) spectroscopy in which a quantum cascade laser is equipped with a highly focused infrared light source to attain a spatial resolution of 100 µm. This system was applied to the forewing of a European hornet to reveal how the protein domains are organised. Two-dimensional patterns were obtained from the VCD signals with steps of 100 µm. We scanned the 1500-1740 cm-1 wavenumber range, which covers amide I and II absorptions. Zone sequenced α-helical and ß-sheet domains within an area of 200 µm2 in membranes close to where two veins cross. The sign of the VCD signal at 1650 cm-1 changed from positive to negative when probed along the zone axis, intermediated by the absence of VCD activity. The significance of this zone is discussed from the viewpoint of the mechanical properties required for flying motion. These features are unattainable using conventional FTIR (Fourier transform infrared) or FT-VCD methods with a spatial resolution of ∼10 mm2.

Multidimensional vibrational circular dichroism for insect wings: Comparison of species.

This study reports the microscopic measurements of vibrational circular dichroism (VCD) on four different insect wings using a quantum cascade laser VCD system equipped with microscopic scanning capabilities (named multi-dimensional VCD [MultiD-VCD]). Wing samples, including (i) beetle, Anomala albopilosa (female), (ii) European hornet, Verspa crabro flavofasciata Cameron, 1903 (female), (iii) tiny dragonfly, Nannophya pygmae Rambur, 1842 (male), and (iv) dragonfly, Symetrum gracile Oguma, 1915 (male), were used in this study. Two-dimensional patterns of VCD signals (~10 mm × 10 mm) were obtained at a spatial resolution of 100 µm. Measurements covered the absorption peaks assigned to amides I and II in the range of 1500-1740 cm-1 . The measurements were based on the enhancement of VCD signals for the stereoregular linkage of peptide groups. The patterns were remarkably dependent on the species. In samples (i) and (ii), the wings comprised segregated domains of protein aggregates of different secondary structures. The size of each microdomain was approximately 100 µm. In contrast, no clear VCD spectra were detected in samples (iii) and (iv). One possible reason was that the chain of stereoregular polypeptides was too short to achieve VCD enhancement in samples (iii) and (iv). Notably, the unique features were only observed in the VCD spectra because the IR spectra were nearly the same among the species. The VCD results hinted at the connection of protein microscopic structures with the wing flapping mechanisms of each species.

Evaluating the Stability of Cellulose Nanofiber Pickering Emulsions Using MRI and Relaxometry.

Magnetic resonance imaging (MRI) relaxometry and diffusion methods were used to highlight the instability mechanisms of oil-in-water Pickering emulsions stabilized by cellulose nanofibers (CNFs). Four different Pickering emulsions using different oils (n-dodecane and olive oil) and concentrations of CNFs (0.5 and 1.0 wt %) were systematically investigated over a period of one month after emulsification. The separation into a free oil, emulsion layer, and serum layer and the distribution of flocculated/coalesced oil droplets in several hundred micrometers were captured in MR images using fast low-angle shot (FLASH) and rapid acquisition with relaxation enhancement (RARE) sequences. The components of the Pickering emulsions (e.g., free oil, emulsion layer, oil droplets, and serum layer) were observable by different voxelwise relaxation times and apparent diffusion coefficients (ADCs) and reconstructing in the apparent T1, T2, and ADC maps. The mean T1, T2, and ADC of the free oil and serum layer corresponded well with MRI results for pure oils and water, respectively. Comparing the relaxation properties and translational diffusion coefficients of pure dodecane and olive oil obtained from NMR and MRI resulted in similar T1 and ADC but significantly different T2 depending on the sequence used. The diffusion coefficients of olive oil measured by NMR were much slower than dodecane. The ADC of the emulsion layer for dodecane emulsions did not correlate with the viscosity of the emulsions as the CNF concentration increased, suggesting the effects of restricted diffusion of oil/water molecules due to droplet packing.

Synthetic Nanocelluloses Fluorescently Responsible to Enzymatic Degradation.

Crystalline assemblies of cellulose and cellulose derivatives that can be synthetically produced by in vitro enzymatic reactions in a bottom-up manner have attracted increasing attention as chemically designable functional nanomaterials (e.g., synthetic nanocelluloses). In this study, we demonstrate the preparation and characterization of alkyl ß-celluloside assemblies loaded with fluorescent molecules, which are fluorescently responsible to the enzymatic degradation of the cellulose moieties. The fluorescent properties are afforded to the assemblies by their bilayer-structured nanosheet morphologies realized through the uptake of environmentally responsive fluorescent molecules (namely, Nile Red (NR)). Incubation of the NR-loaded n-octyl ß-celluloside (CEL-C8) assembly with cellulase resulted in decreases in the fluorescence intensities. This suggests that NR molecules were released into the aqueous phase through enzymatic degradation of the cellulose moieties of CEL-C8 molecules in the assembly. The fluorescence decrease rates were clearly dependent on the concentration and source of cellulase. Fluorescence decreases through enzymatic degradation were again observed in the presence of contaminant proteins. These observations revealed the high potential of alkyl ß-celluloside assemblies loaded with fluorescent molecules as fluorescently responsible cellulase substrates for cellulase detection assays by simply measuring changes in the fluorescence intensities. Moreover, the assemblies were revealed as carriers for the controlled release of loaded molecules triggered by enzymatic degradation.

A hybrid strategy combining solution NMR spectroscopy and isothermal titration calorimetry to characterize protein-nanodisc interaction.

NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.

Multidimensional Vibrational Circular Dichroism Apparatus Equipped with Quantum Cascade Laser and Its Use for Investigating Some Peptide Systems Containing d-Amino Acids.

We developed a multidimensional vibrational circular dichroism system with a positional coordinate, making it possible to move in both x- and y-directions using an automatic stage. Quantum cascade laser (QCL) was used as a light source to achieve high intensity and narrow focusing. The QCL emitted light in the wavenumber range of 1500-1740 cm-1, which encompassed absorption bands assigned to the stretching vibrations of amides I and II. The operation of the instrument was analyzed for samples containing amide groups. An aqueous solution of Gly-l-Leu or Gly-d-Leu was measured under the background absorbance as high as 3.5 due to the water medium. The spectra were recorded by scanning at 1.0 cm-1 steps. The time required for performing measurement at each wavenumber was less than 1 s. The mirror-image relation was maintained between the optical antipodes. A peak assigned to amide II appeared clearly at around 1580 cm-1. In the case of KBr pellets containing the same compounds, peaks assigned to amide I and II were observed. For two-dimensional pattering, a KBr pellet comprising two domains of amino acids (or l-Ala and d-Ser) was investigated. The distribution of each component within the pellet was obtained under the two-dimensional alignment at the spatial interval of 2.5 mm.

On-Demand Chirality Transfer of Human Serum Albumin to Bis(thiophen-2-yl)hexafluorocyclopentenes through Their Photochromic Ring Closure.

Photochromic 1,2-bis(5-carboxy-3-methyl-2-thienyl)hexafluorocyclopentene and its dimethyl ester incorporated in human serum albumin (HSA) showed highly enantioselective photochromic ring-closing reactions upon 366 nm light irradiation. The absolute stereochemistry of the major ring-closed form of the dicarboxylic acid at the newly formed sp3 carbon atoms was determined to be (S,S) by the process of docking simulation of the diarylethene molecule and HSA followed by molecular dynamics calculations and comparison of the measured and calculated CD spectra. Esterification of the major closed form of the diacid gave the minor closed form of the diester. The absolute stereochemistry of the major diester was thus determined to be (R,R).

Vibrational circular dichroism of d-amino acid-containing peptide NdWFamide in the crystal form.

Microcrystals of l-Asn-d-Trp-l-Phe-NH2 (NdWFamide), a tripeptide derived from Aplysia kurodai that exhibits invertebrate cardiac activity, were evaluated by vibrational circular dichroism (VCD). The chirality of the tryptophan residue at the second position in NdWFamide was associated with the conformation and biological characteristics. The VCD spectrum of NdWFamide was a mirror image of its enantiomer; however, it was significantly different from that of its diastereomer, NWFamide, which is its precursor. The obtained VCD signals of NdWFamide were in good agreement with the VCD signals that were calculated based on the optimized aggregates of NdWFamide, which formed a helical-like backbone conformation. The evaluation of the VCD results revealed the conformation of NdWFamide in the crystalline state and succeeded in distinguishing its stereoisomers. Therefore, this study demonstrates VCD as a useful method for the structural analysis of naturally occurring d-amino acid-containing peptides.

Rheological and Ionic Transport Properties of Nanocomposite Electrolytes Based on Protic Ionic Liquids and Silica Nanoparticles.

In this study, the effect of hydrophilic silica nanoparticle (AEROSIL 200) addition on the rheological and transport properties of several protic ionic liquids (PILs) consisting of protonated 1,8-diazabicyclo[5.4.0]undec-7-ene cation (DBU) was studied. Interactions between the surface silanol groups of the silica nanoparticles and the ions of these PILs affected the nature of particle aggregation and the hydrogen bonding environment, which was reflected in the nonlinear rheological behaviors and transport properties of their colloidal suspensions. In contrast to shear-thinning gels formed by colloidal suspensions of the silica nanoparticles in [DBU][TFSA] ([TFSA] = [N(SO2CF3)2]), [DBU][TfO] ([TfO] = [CF3SO3]), and [DBU][TFA] ([TFA] = [CF3CO2]), a shear-thickening stable suspension was formed in the [DBU][MSA] ([MSA] = [CH3SO3]) system. A relatively strong interaction between the silanol groups and the ions of [DBU][MSA] and the ability of this PIL to form a thicker solvation layer through hydrogen bonding were assumed to be responsible for this unique behavior. Moreover, the [DBU][MSA]-silica system showed a large enhancement in the conductivity at a certain silica concentration. This enhancement was not observed in the other PIL-silica composites that exhibited shear-thinning behavior. Even though diffusion of ions was found to be restricted in the presence of silica, a preferentially stronger interaction between [MSA] anions and the silica surface resulted in an increase in the number of charge carriers.

Solid-state vibrational circular dichroism studies of L- and D-serine.

d-Serine is considered a key endogenous substance involved in several enzymatic reactions in the human body. In this study, solid-state vibrational circular dichroism (VCD) measurements of enantiomeric serine were performed for spectroscopic distinction between d- and l-serine. The mirror-image VCD signals of the isomers in the KBr pellets were observed in the mid-infrared range of 1800-1250 cm-1. The calculated infrared (IR) and VCD spectra for the optimized serine structures were in good agreement with the corresponding observed spectra. In addition, the chemical shift values estimated from the shielding constants of the optimized structure of serine corresponded with the observed values in 13C and 15N solid-state nuclear magnetic resonance spectra, supporting the VCD assignments. Our results suggest the need for further study of VCD to develop a sensitive and high-resolution spectroscopic technique for the detection of d-amino acids.

Self-assembly of tripeptides into γ-turn nanostructures.

Self-assembling phenylalanine-based peptides have garnered interest owing to their potential for creating new functional materials. Here, we designed four diastereomers, l-Phe-l-Phe-l-Phe (FFF), d-Phe-l-Phe-l-Phe (fFF), l-Phe-d-Phe-l-Phe (FfF) and l-Phe-l-Phe-d-Phe (FFf), to analyze the effect of the d-isomer on the self-assembly. Using SEM, TG, VCD, and solid-state NMR measurements, we found that only FFf forms a γ-turn conformation and self-assembles into a nanoplate with higher thermal stability. The supramolecular structure of FFf consists of intra- and intermolecular hydrogen bonds and π-π stackings. From our results, we have discovered that FFf forms a new type of self-assembling γ-turn conformation, clarifying the structural role of a d-amino acid residue in supramolecular formation.

Solid-state NMR characterization of triacylglycerol and polysaccharides in coffee beans.

It is important to understand the structural characteristics of triacylglycerol (TAG), polysaccharides and trace elements in coffee beans, so that residues can be reutilized in applications including biodiesel oils. Here, we performed 1H and 13C solid-state NMR measurements on Indonesian green beans, roasted beans, and spent coffee grounds (SCGs). In the NMR spectra, there were liquid-like TAG containing linoleic acids based on observed signals of -CH=CH-CH2-CH=CH- group in an acyl chain, which play a role in decreasing TAG's melting point. We found TAG was still abundant in the SCGs from NMR spectra. After lipids were removed from SCGs, the intensity of the TAG signal decreased considerably, with approximately 64% of the TAG was successfully extracted. We described the chemical structure of TAG in coffee beans and demonstrated that it is possible quantify the amount of extracted TAG using solid-state NMR.

Retinal Configuration of ppR Intermediates Revealed by Photoirradiation Solid-State NMR and DFT.

Pharanois phoborhodopsin (ppR) from Natronomonas pharaonis is a transmembrane photoreceptor protein involved in negative phototaxis. Structural changes in ppR triggered by photoisomerization of the retinal chromophore are transmitted to its cognate transducer protein (pHtrII) through a cyclic photoreaction pathway involving several photointermediates. This pathway is called the photocycle. It is important to understand the detailed configurational changes of retinal during the photocycle. We previously observed one of the photointermediates (M-intermediates) by in situ photoirradiation solid-state NMR experiments. In this study, we further observed the 13C cross-polarization magic-angle-spinning NMR signals of late photointermediates such as O- and N'-intermediates by illumination with green light (520 nm). Under blue-light (365 nm) irradiation of the M-intermediates, 13C cross-polarization magic-angle-spinning NMR signals of 14- and 20-13C-labeled retinal in the O-intermediate appeared at 115.4 and 16.4 ppm and were assigned to the 13-trans, 15-syn configuration. The signals caused by the N'-intermediate appeared at 115.4 and 23.9 ppm and were assigned to the 13-cis configuration, and they were in an equilibrium state with the O-intermediate during thermal decay of the M-intermediates at -60°C. Thus, photoirradiation NMR studies revealed the photoreaction pathways from the M- to O-intermediates and the equilibrium state between the N'- and O-intermediate. Further, we evaluated the detailed retinal configurations in the O- and N'-intermediates by performing a density functional theory chemical shift calculation. The results showed that the N'-intermediate has a 63° twisted retinal state due to the 13-cis configuration. The retinal configurations of the O- and N'-intermediates were determined to be 13-trans, 15-syn, and 13-cis, respectively, based on the chemical shift values of [20-13C] and [14-13C] retinal obtained by photoirradiation solid-state NMR and density functional theory calculation.

The role of d-allo-isoleucine in the deposition of the anti-Leishmania peptide bombinin H4 as revealed by 31P solid-state NMR, VCD spectroscopy, and MD simulation.

Bombinin H4 is an antimicrobial peptide that was isolated from the toad Bombina variegata. Bombinin H family peptides are active against gram-positive, gram-negative bacteria, and fungi as well as the parasite Leishmania. Among them, bombinin H4 (H4), which contains d-allo-isoleucine (d-allo-Ile) as the second residue in its sequence, is the most active, and its l-isomer is bombinin H2 (H2). H4 has a significantly lower LC50 than H2 against Leishmania. However, the atomic-level mechanism of the membrane interaction and higher activity of H4 has not been clarified. In this work, we investigated the behavior of the conformations and interactions of H2 and H4 with the Leishmania membrane using 31P solid-state nuclear magnetic resonance (NMR), vibrational circular dichroism (VCD) spectroscopy, and molecular dynamics (MD) simulations. The generation of isotropic 31P NMR signals depending on the peptide concentration indicated the abilities of H2 and H4 to exert antimicrobial activity via membrane disruption. The VCD experiment and density functional theory calculation confirmed the different stability and conformations of the N-termini of H2 and H4. MD simulations revealed that the N-terminus of H4 is more stable than that of H2 in the membrane, in line with the VCD experiment data. VCD and MD analyses demonstrated that the first l-Ile and second d-allo-Ile of H4 tend to take a cis conformation. These residues function as an anchor and facilitate the easy winding of the helical conformation of H4 in the membrane. It may assist to quickly reach to the threshold concentration of H4 on the Leishmania membrane. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

Long-distance perturbation on Schiff base-counterion interactions by His30 and the extracellular Na+-binding site in Krokinobacter rhodopsin 2.

Krokinobacter rhodopsin 2 (KR2), a light-driven Na+ pump, is a dual-functional protein, pumping protons in the absence of Na+ when K+ or larger alkali metal ions are present. A specific mutation in helix A near the extracellular Na+ binding site, H30A, eliminates its proton pumping ability. We induced structural changes in H30A by altering the alkali metal ion bound at the extracellular binding site, and observed a strong electrostatic interaction between the Schiff base and counterion and torsion around the Schiff base as revealed by solid-state nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies. The strong interaction when His30 was absent and no ion bound at the extracellular binding site disabled retinal reisomerization, as was shown with flash-photolysis, forming a small amount of only a K-like intermediate. This revealed why H30A lacks the proton pumping function. Long-distance perturbation of the binding site and Schiff base revealed that a non-transported ion binding at the extracellular site is essential for pumping.

Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin.

The recently identified Krokinobacter rhodopsin 2 (KR2) functions as a light-driven sodium ion pump. The structure of the retinal-binding pocket of KR2 offers important insights into the mechanisms of KR2, which has motif of Asn112, Asp116, and Gln123 (NDQ) that is common among sodium ion pump rhodopsins but is unique among other microbial rhodopsins. Here we present solid-state nuclear magnetic resonance (NMR) characterization of retinal and functionally important residues in the vicinity of retinal in the ground state. We assigned chemical shifts of retinal C14 and C20 atoms, and Tyr218Cζ, Lys255Cε, and the protonated Schiff base of KR2 in lipid environments at acidic and neutral pH. 15N NMR signals of the protonated Schiff base showed a twist around the N-Cε bond under neutral conditions, compared with other microbial rhodopsins. These data indicated that the location of the counterion Asp116 is one helical pitch toward the cytoplasmic side. In acidic environments, the 15N Schiff base signal was shifted to a lower field, indicating that protonation of Asp116 induces reorientation during interactions between the Schiff base and Asp116. In addition, the Tyr218 residue in the vicinity of retinal formed a weak hydrogen bond with Asp251, a temporary Na+-binding site during the photocycle. These features may indicate unique mechanisms of sodium ion pumps.

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.

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.

Magnetically Alignable Bicelles with Unprecedented Stability Using Tunable Surfactants Derived from Cholic Acid.

Five novel surfactants were prepared by modifying the three hydroxy groups of sodium cholate with triethylene glycol chains endcapped with an amide (SC-C1 , SC-n C4 , and SC-n C5 ) or a carbamoyl group (SC-On C4 and SC-Ot C4 ). The phase behavior of aqueous mixtures of these surfactants with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) was systematically studied by 31 P NMR spectroscopy. The surfactants endcapped with carbamate groups (SC-On C4 and SC-Ot C4 ) formed magnetically alignable bicelles over unprecedentedly wide ranges of conditions, in terms of temperature (from 21-23 to >90 °C), lipid/surfactant ratio (from 5 to 8), total lipid content (5-20 wt %), and lipid type [DMPC, 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)]. In conjunction with appropriate phospholipids, the carbamate-endcapped surfactants afforded unique bicelles, characterized by exceptional thermal stabilities (from 0 to >90 °C), biomimetic lipid compositions (DMPC/POPC=25:75 to 50:50), and extremely large 2 H quadrupole splittings (up to 71 Hz).

Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy.

Photo-reaction pathways of a bacteriorhodopsin Y185F mutant were examined using in situ photo-irradiation solid-state NMR spectroscopy. (13)C CP MAS NMR spectra were recorded at -40 °C in the dark (D1), under irradiation with 520 nm light (L1), subsequently in the dark (D2), and again under irradiation with 520 nm light (L2). In the process from D1 to L1, the 13-cis, 15-syn (CS; bR548) state changed to a CS*- (13-cis, 15-syn) intermediate, which was highly stable at -40 °C, and the all-trans (AT; bR568) state transformed to an N-intermediate. Under the D2 conditions, the N-intermediate transformed to an O-intermediate, which was highly stable at -40 °C in the dark. During subsequent irradiation with 520 nm light (L2), the O-intermediate transformed to the N-intermediate through the AT state, whereas the CS*-intermediate did not change. The CS*-intermediate was converted to the AT state (or O-intermediate) after the temperature was increased to -20 °C. Upon subsequent increase of the temperature to 20 °C, the AT state (or O-intermediate) was converted to the CS state until reaching equilibrium. In this experiment, the chemical shift values of [20-(13)C, 14-(13)C]retinal provided the 13C[double bond, length as m-dash]C and 15C[double bond, length as m-dash]N configurations, respectively. From these data, the configurations of the AT and CS states and the CS*-, N-, and O-intermediates were determined to be (13-trans, 15-anti), (13-cis, 15-syn), (13-cis, 15-syn), (13-cis, 15-anti), and (13-trans, 15-anti), respectively. (13)C NMR signals of the CS*- and O-intermediates were observed for the first time for the Y185F bR mutant by in situ photo-irradiation solid-state NMR spectroscopy and the configuration of the CS*-intermediate was revealed to be significantly twisted from that of the CS state although both were assigned as (13-cis, 15-syn) configurations.