Masashi Sonoyama

SOSUI-GramN: high performance prediction for sub-cellular localization of proteins in gram-negative bacteria.

A predictive software system, SOSUI-GramN, was developed for assessing the subcellular localization of proteins in Gram-negative bacteria. The system does not require the sequence homology data of any known sequences; instead, it uses only physicochemical parameters of the N- and C-terminal signal sequences, and the total sequence. The precision of the prediction system for subcellular localization to extracellular, outer membrane, periplasm, inner membrane and cytoplasmic medium was 92.3%, 89.4%, 86.4%, 97.5% and 93.5%, respectively, with corresponding recall rates of 70.3%, 87.5%, 76.0%, 97.5% and 88.4%, respectively. The overall performance for precision and recall obtained using this method was 92.9% and 86.7%, respectively. The comparison of performance of SOSUI-GramN with that of other methods showed the performance of prediction for extracellular proteins, as well as inner and outer membrane proteins, was either superior or equivalent to that obtained with other systems. SOSUI-GramN particularly improved the accuracy for predictions of extracellular proteins which is an area of weakness common to the other methods.

Proportion of membrane proteins in proteomes of 15 single-cell organisms analyzed by the SOSUI prediction system.

A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378-379). The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices. When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15-20%, irrespective of the total genome size. However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.

Second Derivative near Infrared Studies on the Structural Characterisation of Proteins

Fourier Tranform near infrared (FT-NIR) spectra have been measured for several globular proteins which vary widely in the relative amounts of different secondary structures. In order to assist in the interpretation of the NIR spectra in the region between 5000 and 4000 cm−1, FT-NIR measurements of silk fibroin with different secondary structures were also carried out. The second derivative procedures showed that the broad contour near 4610 cm−1 consists of several overlapping components and each relative intensity is significantly changed with the proteins. In particular, the band near 4525 cm−1 seemed to be a characteristic component of sheet structure. On the other hand, low-wavenumber NIR bands in 4500– 4000 cm−1 region were considered to reflect the amino acid compositions of proteins. These experimental data suggested that NIR spectroscopy can be used for structural analyses and characterisations of proteins.

Infrared Rheo-Optics of Bombyx Mori Fibroin Film by Dynamic Step-Scan FT-IR Spectroscopy Combined with Digital Signal Processing

Recently developed dynamic step-scan FT-IR spectroscopy combined with software-based digital signal processing (DSP) was applied for infrared rheo-optical measurements of isotactic polypropylene and regenerated Bombyx mori silk fibroin film undergoing sinusoidal mechanical strain. Comparative measurements of dynamic infrared spectra of polypropylene film using the DSP-based method and two lock-in amplifiers (LIAs) indicated that a high signal-to-noise ratio (SNR) advantage is attained by the DSP method compared with the LIA method. With the DSP technique, dynamic spectra of Bombyx mori silk fibroin film with high SNR were successfully recorded in the whole mid-infrared region in only 30 min. The dynamic spectra revealed that stress-induced dynamic reorientation in fibroin film is mainly localized in the segment with β-sheet conformation and is almost synchronous with the applied mechanical strain. Two-dimensional (2D) correlation analyses of the dynamic spectra showed that (1) the broad amide I band is resolved into three components whose positions are dependent on secondary structures, (2) the dynamic behavior of tyrosine residues was extracted by separation of the feature due to its aromatic side chains from the broad envelope of the amide I band in the 2D asynchronous map, and (3) the dynamic behavior of the amide A and B modes is the same as that of the amide II mode, when the mechanical strain is applied to fibroin film.

Characterization of water contribution to excimer laser ablation of collagen

Abstract In order to gain an obvious insight into the role of water in the mechanism of the excimer laser ablation of the cornea, we have macroscopically investigated the ablation behavior of collagen gel in the swelled state by direct photoetching using an ArF excimer laser with time-resolved photography, and furthermore, the thermal effects on the microscopic structures of the collagen molecules by FTIR–ATR spectroscopy. The hydrated collagen film (HF) has a smaller threshold fluence than the dried collagen film (DF). From the time-resolved photographs, the ejected materials were detected only for HF. It was predicted that the effect of bubble formation for HF contributes to the etching. The FTIR–ATR spectroscopic results revealed that the existence of the water suppressed the denaturation of the collagen to gelatin on the surface in the irradiated region. Overall, it was inferred that during the ablation process for HF, the laser energy would be mostly consumed as the latent heat of evaporation of water, that is, the water in the gel matrix would contribute to the suppression of the increment in the temperature in the irradiated region.

Dynamic Step-Scan Two-Dimensional Fourier Transform Infrared Studies of Uniaxially Drawn Poly(ethylene terephthalate) Film

Dynamic infrared spectra of uniaxially drawn poly(ethylene terephthalate) (PET) under a sinusoidal strain were examined. A very intense dynamic band at 973 cm−1 assigned to the trans C-O stretching mode indicated stress-induced high mobility around the C-O bond in the ethylene glycol units. It was supposed that derivative-like skeletal bands observed in the dynamic spectra originated from the stress-induced frequency shift. Two-dimensional correlation analyses of the dynamic spectra were also carried out and revealed that the phenyl ring 18a band at 1018 cm−1 and the phenyl ring 19b band at 1410 cm−1 were composed of three and two independent components, respectively. The correlation peaks between the phenyl ring and CH2 vibrational modes showed that orientation of the methylene group in the ethylene glycol unit, induced by mechanical stretching, is faster than that of the phenyl ring in the terephthalate unit.

Dynamic FT-IR Spectroscopic Studies of Silk Fibroin Films

Dynamic FT-IR spectra in the amide I region were measured for silk fibroin cast films. Phase analyses of the dynamic spectra were carried out in order to separate them into several components which are due to the secondary structures of fibroin. These observations showed that peak positions of the amide I bands were in good agreement with those obtained by Fourier self-deconvolution procedures. In conclusion, phase analysis of dynamic FT-IR spectra is a powerful technique for separation of highly overlapping bands.

Secondary structure and thermostability of the photosystem II manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus

The secondary structure of the manganese-stabilizing protein of the thermophilic cyanobacterium Synechococcus elongatus in solution was investigated by Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopies. Both methods showed a high proportion of disordered structure (40-43%) and a relatively small amount of beta-sheet (23-24%) and alpha-helix (17-19%). The conformation of the protein remained essentially unchanged at temperatures up to 70 degrees C. Unfolding of the protein occurred at higher temperatures and FT-IR spectroscopy revealed that beta-sheet was more strongly unfolded than alpha-helix at 76 degrees C. The protein largely lost the ordered secondary structures at 90 degrees C, but, when cooled down to 30 degrees C, regained its original conformation. Thus, the cyanobacterial protein is very thermostable and its denaturation at an extremely high temperature is reversible.

Photobleaching of Bacteriorhodopsin Solubilized with Triton X‐100

Abstract In the current studies, we examined the effects of hexagonal lattice formation with lipid membranes on the structural stability of native bacteriorhodopsin (bR). Denaturation kinetic measurements for bR solubilized with the mild nonionic detergent Triton X-100 (TX100) were performed in the dark and under illumination by visible light. The solubilized bR was stable in the dark over a wide concentration range of TX100 (1 to 200 mM). In purple membranes, a bilobed band was observed in visible circular dichroism spectra due to interactions between neighboring chromophores. At all concentrations of TX100, this was replaced by a single positive band. Upon illumination with visible light, TX100-solubilized bR clearly showed photobleaching to bacterioopsin. These experimental results suggest that photobleaching is due to a lack of intermolecular interactions inside the purple membrane lattice. Extensive kinetic measurements further revealed that the rate constant of photobleaching is strongly dependent on the detergent concentration, although the activation energy for photobleaching does not significantly change with the TX100 concentration. The mechanism of photobleaching for the solubilized bR is discussed with respect to detergent micelle properties.

Inhomogeneous stability of bacteriorhodopsin in purple membrane against photobleaching at high temperature

Heterogeneity in the state of bacteriorhodopsin in purple membrane was studied through temperature jump experiments carried out in darkness and under illumination with visible light. The thermal denaturation, the irreversible component of spectral change at high temperature, had two decay components, suggesting that bacteriorhodopsin in purple membrane has heterogeneous stability. The temperature dependence of kinetic parameters under illumination revealed that the fast‐decay component gradually increased at above 60°C, indicating that the proportion of unstable bacteriorhodopsin increased. Significant change in the visible circular dichroism (CD) spectra was observed in darkness in the same temperature range as the increase of the fast‐decay component under illumination. Denaturation experiments for C‐terminal‐cleaved bacteriorhodopsin showed that the C‐terminal segment had some effect on the structural stability of bacteriorhodopsin under illumination. Dynamic and static models of the inhomogeneous stability of bacteriorhodopsin in purple membrane are discussed on the basis of the results of the denaturation kinetics and the visible CD spectra. Proteins 2004;54:000–000.