Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs.
Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET-FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.
Visser
A J W G
AJ
Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET, Wageningen, The Netherlands. antoniejvisser@gmail.com
Laptenok
S P
SP
Visser
N V
NV
van Hoek
A
A
Birch
D J S
DJ
Brochon
J-C
JC
Borst
J W
JW
D000465:Algorithms; D001419:Bacteria; D001426:Bacterial Proteins / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D008164:Luminescent Proteins; C527701:enhanced cyan fluorescent protein; C485184:enhanced green fluorescent protein; C495639:yellow cameleon; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium
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