The formation and structure of the potassium complex with valinomycin in solution were studied by means of Raman and Raman optical activity (ROA) spectroscopy. The complexation caused significant spectral changes, particularly in the region 1200-1400 cm(-1). The experimental spectra were interpreted using first principles computations. A complete computational conformational search combined with the spectral analysis revealed the arrangement of the isopropyl side chains in the complex. From a total of 6579 unique conformers two predominant ones were confirmed in the solution by ROA. A third one was predicted theoretically, but its population in the experiment could be estimated only roughly. The most populated conformer does not exhibit C(3) symmetry, and is different from that present in the crystal and the NMR-derived structure. Molecular dynamics techniques were used to estimate the molecular flexibility and its effect on the spectra. Density functional computations and Cartesian coordinate transfer (CCT) techniques provided the ROA and Raman spectral shapes and intensities well comparable with the experiment. The polar solvent (methanol) environment modeled with a polarizable continuum model (PCM) leads to rather minor changes in the conformer populations and vibrational properties as compared to vacuum computations, due to the hydrophobic character of the complex. Additional computational experiments suggest that the vibrational interactions determining the ROA spectra are quite local, which contributes to the good spatial resolution of the method. A reduction of the noise in the experimental spectra as well as increased precision of the simulations is desirable for the further exploration of the potential of the ROA spectroscopy for biomolecular studies in the future.

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å . The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ␤-pore-forming toxins such as aerolysin, C. perfringens ⑀-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ␤-strands, two of which span its entire length. Domain II and domain III have three short ␤-strands each, by which they are distinguished. In addition, domain II has an ␣-helix lying on the ␤-strands. The sequence of amino acids composing the ␣-helix and preceding ␤-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ␤-barrel-made pores. These structural features imply that CPE is a ␤-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ␤-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins. Clostridium perfringens enterotoxin (CPE),2 which damages intestinal epithelia, is a causative agent of food poisoning. The toxin consists of a single chain polypeptide of 319 amino acids. The C-terminal domain of CPE (C-CPE, residues 184 -319) recognizes and binds to certain members of the claudin family, components of tight junctions, as a receptor on target cells (1-4), and the N-terminal region is believed to be involved in forming physiological pores to disrupt the selective permeability of the plasma membrane, resulting in cell death (5-7). It was also reported that the physiological pores are composed of a large complex comprising CPE and cellular components such as claudins (2,8).The binding between C-CPE and claudins has been well characterized. The 16 -17 C-terminal amino acids of C-CPE were reportedly important for the interaction (9, 10), especially, Tyr 306 , Tyr 310 , Tyr 312, and Leu 315 (11-13). According to the crystal structure of C-CPE (14), these residues organize a cleft space that is considered to interact directly with claudins. Claudins are tetratransmembrane proteins. The region of claudins responsible for binding to CPE was located on the C-terminal side of the second extracellular loop and recently designated CPE-SR for CPE sensitivity-related region (15). The bottom of the cleft space of CPE is negatively charged, whereas the CPESRs of CPE-sensitive claudins are positively charged. Therefore, it was proposed that electrostatic attraction at...

Vibrio cholerae is an aetiological agent of cholera that inhabits marine and estuarine environments. It can survive harsh environments by entering the viable but non-culturable (VBNC) state, but the related changes in gene expression have not been described. Here, we experimentally induced the VBNC state in V. cholerae O1, by incubation in artificial seawater at 4°C. Bacterial cells that were incubated for 70 days retained their membrane integrity and were pathogenic, colonizing the gut of iron-dextran-treated mice, even though they formed no colonies on tryptic soy agar (TSA) or TSA amended with pyruvate. We therefore used this stage of cells as the VBNC bacteria. We compared the global transcription pattern of the VBNC cells with that of stationary-phase cells grown in rich medium. A total of 100 genes were induced by more than fivefold in the VBNC state, and the modulated genes were mostly those responsible for cellular processes. Furthermore, real-time RT-PCR analysis verified the changes in the expression levels, showing that the VC0230 [iron(III) ABC transporter], VC1212 (polB), VC2132 (fliG) and VC2187 (flaC) mRNAs were increased in the non-culturable state. Thus, these genes may be suitable markers for the detection of VBNC V. cholerae. To our knowledge, this is the first report of a comprehensive transcriptome analysis of V. cholerae in the VBNC state. The significance of this gene expression profile compared with those of in vivo isolates and non-stressed bacteria (culturable in vitro) is its potential to provide information about the public health risk from dormant bacteria.

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