Possible bi-stable structures of pyrenebutanoic acid-linked protein molecules adsorbed on graphene: theoretical study.

We theoretically analyze possible multiple conformations of protein molecules immobilized by 1-pyrenebutanoic acid succinimidyl ester (PASE) linkers on graphene. The activation barrier between two bi-stable conformations exhibited by PASE is confirmed to be based on the steric hindrance effect between a hydrogen on the pyrene group and a hydrogen on the alkyl group of this molecule. Even after the protein is supplemented, this steric hindrance effect remains if the local structure of the linker consisting of an alkyl group and a pyrene group is maintained. Therefore, it is likely that the kinetic behavior of a protein immobilized with a single PASE linker exhibits an activation barrier-type energy surface between the bi-stable conformations on graphene. We discuss the expected protein sensors when this type of energy surface appears and provide a guideline for improving the sensitivity, especially as an oscillator-type biosensor.

Theoretical Analysis on the Stability of 1-Pyrenebutanoic Acid Succinimidyl Ester Adsorbed on Graphene.

The adsorbed structure of 1-pyrenebutanoic acid succinimidyl ester (PASE) on graphene was investigated based on density functional theory. We found two locally stable structures: a straight structure with the chainlike part of butanoic acid succinimidyl ester (BSE) lying down and a bent structure with the BSE part directed away from graphene, keeping the pyrene (Py) part adsorbed on graphene. Then, to elucidate the adsorption mechanism, we separately estimated the contributions of the Py and BSE parts to the entire PASE adsorption, and the adsorption effect of the BSE part was found to be secondary in comparison to the contribution of the Py. Next, the mobility of the BSE part at room temperature was confirmed by the activation energy barrier between straight and bent structures. To take account of the external environment, we considered the presence of amino acids and the hydration effect by a three-dimensional reference interaction site model. The contributions of glycine molecules and the solvent environment to stabilizing the bent PASE structure relative to the straight PASE structure were found. Therefore, the effect of the external environment around PASE is of importance when the standing-up process of the BSE part from graphene is considered.

Efficacy of a solar-powered TiO2 semiconductor electric toothbrush on P. gingivalis biofilm.

PURPOSE: To determine the efficacy of a solar-powered TiO2 semiconductor electric toothbrush on Porphyromonas gingivalis biofilm. METHODS: P. gingivalis cells were cultivated on sterilized coverslips under anaerobic conditions and were used as a biofilm. To evaluate the efficacy of the solar-powered TiO2 electric toothbrush on the P. gingivalis biofilm, the bacterial cell biofilm coverslips were placed into sterilized phosphate buffered saline (PBS) and brushed for 1 minute. Following mechanical brushing, the coverslips were stained with 1% crystal violet (CV) for 10 seconds at room temperature. The efficacy of P. gingivalis biofilm removal by the solar-powered TiO2 electric toothbrush was measured through the absorbance of the CV-stained solution containing the removed biofilm at 595 nm. The antimicrobial effect of the solar-powered TiO2 semiconductor was evaluated by the P. gingivalis bacterial count in PBS by blacklight irradiation for 0 to 60 minutes at a distance of 7 cm. The electrical current though the solar-powered TiO2 semiconductor was measured by a digital multimeter. The biofilm removal by the solar-powered TiO2 semiconductor was also evaluated by scanning electron microscopy (SEM). RESULTS: The biofilm removal rate of the solar-powered TiO2 electric toothbrush was 90.1 ± 1.4%, which was 1.3-fold greater than that of non-solar-powered electric toothbrushes. The solar-powered TiO2 semiconductor significantly decreased P. gingivalis cells and biofilm microbial activity in a time-dependent manner (P< 0.01). The electrical current passing through the solar-powered TiO2 semiconductor was 70.5 ± 0.1 µA, which was a 27-fold higher intensity than the non-solar-powered brush. SEM analysis revealed that the solar-powered TiO2 semiconductor caused a biofilm disruption and that cytoplasmic contents were released from the microbial cells.

Purification and characterization of a novel secondary fimbrial protein from Porphyromonas gulae.

BACKGROUND: Porphyromonas gulae are black-pigmented anaerobic bacteria isolated from the gingival sulcus of various animal hosts and are distinct from Porphyromonas gingivalis originating in humans. We previously reported the antigenic similarities of 41-kDa fimbriae between P. gulae ATCC 51700 and P. gingivalis ATCC 33277. In this study, to clarify the presence of another type of fimbriae of P. gulae, we have purified and characterized the secondary fimbrial protein from P. gulae ATCC 51700. METHODS: The secondary fimbrial protein was purified from P. gulae ATCC 51700 using an immunoaffinity column coupling with antibodies against the 41-kDa fimbrial protein. The expression of fimbriae on the cell surface of P. gulae ATCC 51700 was investigated by transmission electron microscopy. The N-terminal amino acid sequence was determined by an amino acid sequencer system. RESULTS: The molecular mass of this protein was approximately 53-kDa, as estimated by SDS-PAGE. The polyclonal antibodies against the 53-kDa protein did not react with the 41-kDa fimbrial protein of P. gulae ATCC 51700. Immunogold electron microscopy revealed that anti-53-kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. The amino acid sequence of the N-terminal 15 residues of the 53-kDa fimbrial protein showed only 1 of 15 residues identical to the 41-kDa fimbrial protein. CONCLUSION: The 53-kDa fimbriae are different in molecular weight and antigenicity from the 41-kDa fimbrial protein of P. gulae ATCC 51700. These results clearly suggest that the 41-kDa and the 53-kDa fimbriae are distinct types of fimbriae expressed simultaneously by this organism.

Molecular and antigenic similarities of the fimbrial major components between Porphyromonas gulae and P. gingivalis.

Porphyromonas gulae is black-pigmented anaerobic bacteria associated with canine periodontitis. There is little information available about the specific identify and relative occurrence of pigmented anaerobes in companion animals. Our aim was to clarify the factor involved in the adherence and colonization of the organism in the oral cavity. Fimbrial protein was purified from P. gulae ATCC 51700. The molecular mass of this protein was approximately 41kDa as estimated by SDS-PAGE. An antibody against 41-kDa fimbrial protein from P. gingivalis ATCC 33277 reacted with fimbrillin of P. gulae ATCC 51700. Immunogold electron microscopy revealed that the anti-41kDa fimbrial serum bound to fimbria on the cell surface of P. gulae ATCC 51700. Thus, fimbrial protein of P. gulae ATCC 51700 had the same size and antigenicity as 41-kDa fimbriae of P. gingivalis ATCC 33277. The nucleotide sequence of the fimA gene from P. gulae ATCC 51700 showed 94% homology with that of P. gingivalis ATCC 33277. Moreover, the deduced amino acid sequences have 96.8% identity. P. gulae has adherent ability to gingival epithelial cells. The properties of P. gulae fimbriae are similar to those of P. gingivalis fimbriae. We suggest that the surface structure of P. gulae may play a role in the colonization of this organism in periodontal pockets in companion animals.