Site-specific fluorescence double-labeling of proteins and analysis of structural changes in solution by Fluorescence Resonance Energy Transfer (FRET)

Abstract

Graphical abstract

A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer

“…Assuming that the dipole orientation factor ( κ 2 ) was a limiting value ( κ 2 is taken as 2/3 if the orientation of the donor and acceptor is assumed to be random), n is the refractive index of water (1.333), ϕ D is the quantum yield of GFP (0.68), 24 J is the integral of the overlap of the emission from GFP K25C and the absorption from Cyt b 562 N80C given by: where J was calculated using the experimental spectra giving J = 5.67 × 10 14 M −1 cm −1 nm 4 . 25 …”

“…where J was calculated using the experimental spectra giving J ¼ 5.67 Â 10 14 M −1 cm −1 nm 4 . 25 The apparent distance (r app ) is estimated with the value of energy efficiency E derived from time-resolved uorescence measurement and eqn (5).…”

Applications of fluorescence spectroscopy in protein conformational changes and intermolecular contacts

“…But fluorescence may also come from the in-site formed chromophores such as those with heteroaromatic (imidazolinone) ring typical for fluorescent proteins [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] . The last phenomena are explored with success in fluorescence microscopy [47] , [48] , [49] , [50] , [51] , [52] , [53] , [54] , [55] , [56] , [57] , [58] , [59] , [60] , [61] , [62] , [63] , [64] , [65] , [66] , [67] , [68] , [69] , [70] , [71] , [72] , [73] , [74] , [75] , [76] , [77] , [78] , [79] , [80] , [81] , [82] , [83] , [84] , [85] , [86] , [87] , [88] , [89] , [90] ,…”

Three-dimensional Multicolor Subcellular Imaging by Fast Serial Sectioning Tomography for Centimeter-scale Specimens

“…Over the past decade, optical microscopy assisted with mechanical serial sectioning or tissue clearing has been proven as an automated and registration-free solution for large centimeter-scale 3D imaging [8][9][10][11][12][13] . However, both methods require and propidium iodide (PI)) have been used together for double labeling [41][42][43] , which is helpful to provide high color contrast, hence revealing rich biological information (Supplementary Fig. 1).…”

“…First, conventional fluorescent dyes with different emission spectra can be excited simultaneously with a single low-cost UV-LED, providing a broad and informative color palette. In TRUST, two fluorescent dyes (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) have been used together for double labeling [41][42][43] , which is helpful to provide high color contrast, hence revealing rich biological information (Supplementary Fig. 1).…”