Fluorescence-Based Protein Stability Monitoring—A Review

Abstract

Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein

“…DSF works by monitoring a change in fluorescence emission as a function of temperature. The most common method involves the use of a hydrophobic dye or “label” that fluoresces when bound to unfolded (denatured) protein 26 . Jackson et al used DSF to investigate the binding of a wide range of legacy and emerging PFAS to human serum albumin (HSA) using the dye GloMelt™ as an indicator.…”