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Multidonor deep-UV FRET study of protein-ligand binding and its potential to obtain structure information.

The journal of physical chemistry. B (2011-10-15)
Qiang Li, Stefan Seeger
ABSTRACT

Fluorescence resonance energy transfer (FRET) using biotinylated β-galactosidase (βGAL) as a donor and Alexa Fluor 350 (AF350) labeled avidin as an acceptor has been investigated by means of steady-state fluorescence and time-resolved fluorescence spectroscopy. The donors are readily paired with acceptors through the well-established binding affinity of biotin and avidin. The fluorescence energy transfer efficiency was determined by the donor fluorescence emission and lifetime changes in the presence and absence of acceptor. The theoretical energy transfer efficiency and theoretical average distance between donor and acceptor after noncovalent binding was calculated by taking the distribution of tryptophan residues in βGAL and avidin as well as the location of AF350 in avidin into account, which agree with the experimental data. It is shown how information of the location of the acceptor can be obtained. Further, the fluorescence intensity image of AF350 on a biotinylated βGAL-coated quartz surface through UV FRET has been recorded using deep UV laser-based fluorescence lifetime microscopy. The results demonstrate that (a) deep UV laser-based fluorescence lifetime microscopy is a simple and useful method to study UV FRET of proteins using intrinsic fluorescence, (b) structural information even in complex multidonor systems can be obtained, and (c) FRET signals can be obtained to detect binding events using the native fluorescence of proteins as multidonor systems.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 390 azide, suitable for fluorescence
Supelco
Atto 390 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)

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