Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Abstract

Abstract. Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates

“…A rise in fluorescence quantum yield of small aromatic molecules upon binding to amyloid fibrils is well known from molecules like thioflavin or N-arylaminonaphtalene sulfonates [29][30][31]. For these molecules the increase in fluorescence is assumed to originate from the rigid and/or hydrophobic environment at the binding site of the aggregates [30][31][32].…”

“…For these molecules the increase in fluorescence is assumed to originate from the rigid and/or hydrophobic environment at the binding site of the aggregates [30][31][32]. Although these compounds have been studied extensively, the fluorescence mechanism and the origin of the fluorescence increase upon binding are still controversial [31,[33][34][35]. For the DPPcompounds one may assume that a rigid environment at the binding sites of the fibrillar aggregates [30] could reduce the intrinsic flexibility of the compounds and with that could slow down internal conversion processes.…”

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

“…56 Thermal unfolding of Rituximab was explored using a high-throughput microarray method, 57 in which the fluorescence is detected by a MyiQ single-color real-time PCR detection system (Bio-Rad Labs., Hercules, CA). A 480-nm excitation filter with 40-nm bandwidth and a 540-nm emission filter with 50-nm bandwidth were used.…”

How fluorescent labelling alters the solution behaviour of proteins

“…19 Thus, the use of fluorescent labelling of proteins plays an important role for the exploration and understanding of the mechanisms involved in many biological problems, such as intra/inter-cellular communication, genomics, unravelling the origins of pathologies associated with protein condensation diseases and the mechanisms that govern protein-self association during the production of proteins as therapies. 12,18,20,21 To fluorescently label proteins, there are currently three main established strategies; small organic molecules that are covalently attached to the biomolecule of interest, fusion proteins in which an inherently fluorescent protein is co-expressed (or fused) with the protein of interest or the use of quantum dots (or small molecule fluorescent dyes), that are functionalised to form a biotin-avidin pair with the biomolecule under study. [22][23][24] Of these three routes, covalent attachment of an organic fluorophore is considered to be the most versatile method of labelling for fluorescence imaging and sensing of biological specimens.…”