Search results for "Fluorescence in the life sciences"
showing 4 items of 4 documents
Confocal microscopy of single molecules of the green fluorescent protein
1998
Single molecule detection has been extended into life sciences by use of strongly fluorescent labels. The green fluorescent protein (GFP) as a self-fluorescent biomolecule has attracted considerable attention. Here, single molecules of the GFP-mutant Glu222Gln are immobilized in a polyvinylalcohol matrix and detected by confocal fluorescence microscopy. Although this mutant stabilizes one of both conformers of the wild-type GFP, the investigation of its fluorescence dynamics reveals strong signal fluctuations. This fluorescence behaviour is—at least partly—caused by reversible photochemical changes of the protein framework, that can relax into the fluorescent state on different timescales. …
Spectroscopic Methods for the Determination of Protein Interactions
2005
This unit provides guidelines on how to use steady-state fluorescence spectroscopy for the quantification of protein-protein interactions. The fluorescence of a protein is characterized by its excitation and emission spectra, quantum yield, and anisotropy. These parameters can change upon interaction with another protein and can be used to measure the extent of complex formation. The source of fluorescence can be an intrinsic fluorophore, such as tryptophan or tyrosine; a covalently attached fluorescent dye; or a fluorescent binding partner, such as a nucleotide or cofactor, that interacts specifically with the complex. Protocols are provided in this unit for determining affinity constants …
The oxidation state of a protein observed molecole-by-molecule.
2005
We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectros…
Fluorescence Properties of the Chromophore-Binding Domain of Bacteriophytochrome from Deinococcus radiodurans
2013
Fluorescent proteins are versatile tools for molecular imaging. In this study, we report a detailed analysis of the absorption and fluorescence properties of the chromophore-binding domain from Deinococcus radiodurans and its D207H mutant. Using single photon counting and transient absorption techniques, the average excited state lifetime of both studied systems was about 370 ps. The D207H mutation slightly changed the excited state decay profile but did not have a considerable effect on the average decay time of the system or the shape of the absorption and emission spectra of the biliverdin chromophore. We confirmed that the fluorescence properties of both samples are very similar in vivo…