FRET-sensor with nonfluorescent protein as an acceptor was synthesized to observe caspase-3 activity in lifetime mode.
We inserted caspase-3 cleavable linker between red highly fluorescent protein TagRFP and chromoprotein KFP.
Dynamic light scattering was used to determine size of the fusion protein. Incubation with caspase-3 lead to increase
both fluorescence intensity and lifetime of the construction. Cleavage of the linker between proteins was confirmed by
electrophoresis and immunoblotting. FLIM-microscopy showed the differences between fluorescence decays of A549
cell line expressed TagRFP and TagRFP-23-KFP.
pH-dependent aggregation and dissociation of yellow fluorescent protein zFP538 were studied by gel-filtration, dynamic light scattering, and fluorescence spectroscopy. According to the gel-filtration data for low concentration of zFP538 the molecular weight of aggregates decreases upon changing pH from alkaline to neutral. Dynamic light scattering showed that zFP538 aggregates strongly in concentrated solutions. Aggregation influences heavily the pH-profile of fluorescence of zFP538 and stabilizes zFP538 against fluorescence quenching on acidification. Reduction of the protein concentration results in the shifting of pH profile to the alkaline region. Conclusion: aggregation of the yellow fluorescent protein zFP538 depends on pH; dilution of the protein solution is accompanied by dissociation of zFP538 aggregates under neutral and alkaline pH.
Bioluminescence resonance energy transfer (BRET) is a naturally occurring phenomenon taking place in some marine coelenterates. Emission of light in these organisms involves the energy transfer between chromophores of donor luciferase and acceptor fluorescent protein. Due to the strict dependence of BRET efficiency on the inter-chromophore distance, the phenomenon has been applied to study protein-protein interactions by fusing interacting partners with either donor or acceptor proteins. Here we describe a BRET-based homogeneous protein-protein interaction assay exploiting novel donor-acceptor pair formed by photoproteins of jellyfish Aequorea victoria bioluminescent system, aequorin and green fluorescent protein enhanced variant (EGFP). Two known interacting proteins, streptavidin (SAV) and biotin carboxyl carrier protein (BCCP) were fused, respectively, with aequorin and EGFP. The fusions were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-Aequorin and BCCP-EGFP was followed by BRET between aequorin (donor) and EGFP (acceptor) resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-Aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.
Operation of the low-background IR extrinsic photoconductor detectors and detector arrays depends essentially on the process of spreading of the time-dependent photocurrent. The spreading occurs due to the low non-steady-state screening of electric charges in the detectors. It induces the next non-steady-state affects: the increase in the photoresponse time, the essential difference between the photocurrents in the circuits of the source contact injecting free carriers into the detector and of the contact which serves as a drain for the carriers, and the high cross talk in the detector array. The results of the experimental study of these effects and some other ones in 36- and 48-element linear arrays (Si:Ga) and their theoretical description are presented. The theory is in a satisfactory agreement with the experimental data. Some consequences of these results, which are important for the space-based-astronomy applications of extrinsic detectors, are discussed.
The operation of low-background lR extrinsic photoconductor detectors and detector arrays is essentially dependent on the process of spreading time-dependent photocurrent, which is due to low non-steady-state screening of the electric charges in the detectors. The spreading leads to an increase in the photoresponse time, an essential difference between the non-steady-state photocurrents in the circuits of the source contacts injecting free-charge carriers into the detectors and those in the circuits of the contacts that serve as drains for the carriers, and a high non-steady-state cross talk in the detector arrays. The results of the experimental study of all these effects and some others in 36- and 48-element linear arrays (Si:Ga) as well as their theoretical description are presented. The theory developed is in satisfactory agreement with the experimental data. Some consequences of the results obtained, which are important in space-based astronomy applications of such detectors, are discussed.
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