Spatial Control of Protein Binding on Lipid Bimembrane Using Photoeliminative Linker

Protein adsorption and dissociation on cell membrane surfaces is a topic of important study to reveal biological processes including signal transduction and protein trafficking. We demonstrated here the establishment of a mimic model system for the spatial control of protein adsorption/elimination on a lipid bimembrane using a photochemical technique. The novel photoeliminative linker that we synthesized here consists of three distinct components: a substrate (biotin), a photoeliminative group (4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid), and a lipid bimembrane-adsorbent group (farnesyl). The photoeliminative linker was inserted on the entire surface of the lipid bimembrane and two-dimensionally eliminated by spatial UV irradiation onto the membrane to create a biotin pattern. A target protein, streptavidin was selectively immobilized on the patterned biotin, although it was almost not attached on the nonirradiated region. The streptavidin array was selectively dissociated by UV irradiation onto the entire membrane

Protein Recording Material: Photorecord/Erasable Protein Array Using a UV-Eliminative Linker

Protein patterning on solid surfaces is a topic of significant importance in the fields of biosensors, diagnostic assays, cell adhesion technologies, and biochip microarrays. In this letter, we have established a novel, rapid method for the fabrication of a “protein recording material”, which enables us to spatiotemporally regulate the recording, reading, and erasing of a fluorescent protein array as information by a photochemical technique. A photolinker that we synthesized here was used to control the protein array spatiotemporally. The recording process was almost completed after 1 min of photoirradiation to read a clear pattern consisting of a specific protein−ligand complex with high spatiotemporal resolution. The erasing of the protein array was then achieved by photoirradiation onto the entire patterned surface

A Hydrophilic Azobenzene-Bearing Amino Acid for Photochemical Control of a Restriction Enzyme <i>Bam</i>HI

A novel hydrophilic and negatively charged azobenzene-bearing amino acid, 4‘-carboxyphenylazophenylalanine (azoAla 1), has been designed and synthesized for investigation of the photochemical regulation of the enzyme activity. The properties of photoisomerization and thermal stability of the cis-isomer were similar to those of a commonly used phenylazophenylalanine (azoAla 2). For photochemical control of the enzyme, these two azobenzene-bearing amino acids were incorporated into the specific position at the dimer interface of a restriction enzyme BamHI. These trans-azobenzene derivatives in the BamHI suppressed the enzymatic activity, and the following photoirradiation at 366 nm induced the recovery of its activity. Although the activities of both azoAla-BamHI mutants were same level after a long time irradiation, the recovery of the activity of azoAla 1-BamHI was faster than that of azoAla 2-BamHI with a short time irradiation. This result suggests that the negatively charged carboxylate group introduced into an azobenzene moiety affects the behavior of azoAla in the protein scaffold during the trans−cis photoisomerization

Design and Synthesis of Photochemically Controllable Restriction Endonuclease <i>Bam</i>HI by Manipulating the Salt-Bridge Network in the Dimer Interface

The strategy for the design of photochemically controllable enzymes by manipulating the dimer interface is described. Employing a restriction endonuclease BamHI, the selective incorporation of amino acids having a photoremovable 6-nitroveratryl group into the specific position (Lys132) in the dimer interface of the BamHI mutant (H133A) was performed. The activity of the photofunctionalized BamHI mutant was significantly suppressed, and the following photoirradiation induced the recovery of the activity. In addition, uncaging of the 6-nitroveratryl group introduced to Lys132 did not seriously reduce the catalytic activity and affinity for the substrate. These results indicate that the activity of the enzyme can be effectively regulated by caging and uncaging of the specific amino acid in the dimer interface using the photoremovable group

Detection of the Local Structural Changes in the Dimer Interface of <i>Bam</i>HI Initiated by DNA Binding and Dissociation Using a Solvatochromic Fluorophore

To detect the local structural change in an interface between proteins induced by the substrate binding and dissociation, a solvatochromic fluorescent Nβ-L-alanyl-5-(N,N-dimethylamino)-naphthalene-1-sulfonamide (DanAla) was introduced into 132 position of the dimer interface in BamHI. Before addition of the substrate, the fluorescence from the normal planer excited state of DanAla moiety was observed as a main emission, and thereby the DanAla in the dimer interface is located in the hydrophobic microenvironment. The incubation with the substrate for 20 min induced the gradual increase in fluorescence intensity around 430 nm. The fact reflects that the polarity is reduced by the slight structural change initiated by the formation of the complex with the substrate. Furthermore, the incubation for more than 20 min caused the slight decrease in fluorescence around 430 nm and an appearance of fluorescence (560 nm) due to twisted intramolecular charge transfer (TICT) excited state. Therefore, the DanAla is exposed to comparative polar environment after the dissociation of the substrate. The fluorescence lifetime as a minor component, which is attributed to the TICT excited state, was reduced by addition of the substrate. The results provide that the hydrophobicity in the dimer interface is increased by the substrate binding. Interestingly, we found that the structure of an initial form is different from that of a refolded form after the dissociation of the substrate using a spectral subtraction technique. We have achieved detection of the changing structure induced by the substrate binding and dissociation using a steady-state and time-resolved fluorescence

A Sterically Enforced Bent Form of an Edge-Sharing Dipalladium(II) Complex Attained by a Linked-Bisphosphido Bridge

A Sterically Enforced Bent Form of an Edge-Sharing Dipalladium(II) Complex Attained by a Linked-Bisphosphido Bridg

A Sterically Enforced Bent Form of an Edge-Sharing Dipalladium(II) Complex Attained by a Linked-Bisphosphido Bridge

A Sterically Enforced Bent Form of an Edge-Sharing Dipalladium(II) Complex Attained by a Linked-Bisphosphido Bridg

Identification of PorK, PorN, PorL and PorM proteins from the mutant strains by mass spectrometry (see Fig 8C).

The mascot scores obtained are shown.</p

PorL and PorM form a stable protein complex.

porL/porL’-’myc and wild type P. gingivalis were lysed in DDM and the protein complex associated with PorL-myc was immunoprecipitated using myc agarose. Bound complexes were eluted with either SDS-loading buffer (A), or with myc peptide (B). The eluted complexes were separated by SDS-PAGE and stained with Coomassie blue. The indicated bands in (A) were identified by MS to be 1- PorL and 2-PorM. All the bands in (B) were also identified by MS (Table 2, S5 Table)</p

Cross-linking and mass spectrometry demonstrate interactions between PorK, PorN and PG0189.

Cross-linking and mass spectrometry demonstrate interactions between PorK, PorN and PG0189.</p