Abstract
Förster resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λEX ∼ 280 nm, λEM ∼ 350 nm), in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins.
Highlights
Fluorescence spectroscopy has become a crucial tool in biochemical research by virtue of its robustness, high sensitivity and non-invasiveness [1]
This review aims to provide insight into the utilization of tryptophan (Trp) fluorescence, the dominant source of intrinsic protein fluorescence
Labeling of both the antibodies with either a Förster resonance energy transfer (FRET) donor or acceptor would result in FRET fluorescence upon binding of both antibodies to the protein [71]
Summary
Fluorescence spectroscopy has become a crucial tool in biochemical research by virtue of its robustness, high sensitivity and non-invasiveness [1]. The choice of a fluorophore mainly depends on the photophysical properties (e.g., absorption and emission wavelength, Stokes shift and quantum yield) applicable for specific research purposes and detection techniques Another aspect which may influence the selection of a fluorescent moiety is the ease and selectivity at which it can be integrated in a specific target without impeding the natural function thereof. A brief description of the prevailing methods that utilize the intrinsic fluorescence of Trp residues in (native) target proteins is presented These methods include analysis of Trp fluorescence properties, such as changes in emission wavelength and intensity, absorption maxima and anisotropy, as a result of protein conformational changes. The iFRET technique holds high potential to detect specific target proteins in complex mixtures and even in non-engineered cells
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