Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions

Abstract

Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.

Quinaldine Red as a fluorescent probe for determining the melting temperature (Tm) of proteins: a simple, rapid and high-throughput assay

“…This decline in uorescence following the transition is likely attributed to events occurring at high temperatures such as dissociation of the probe from the complex, rapid protein aggregation/precipitation (resulting in removal of protein from the solution), and the temperature-dependent nature of probe uorescence. 38,44,45 Fig. 1C represents the rst derivative of the melt curve (Fig.…”

“…Notably, QR dye exhibits high stability and is not light-sensitive, distinguishing it from other fluorescent protein-binding dyes such as SYPRO Orange. 31,38…”

“…Notably, QR dye exhibits high stability and is not light-sensitive, distinguishing it from other uorescent protein-binding dyes such as SYPRO Orange. 31,38 In this study, we conducted a detailed investigation to assess the efficacy of Quinaldine Red in determining the melting temperature (T m ) of proteins. The examination involved the evaluation of T m for ve distinct proteins: alcohol dehydrogenase (ADH), carbonic anhydrase (CA), recombinase-A (RecA), thyroglobulin (TG), and human translin (HTsn), based on the differential binding of the uorescent dye (QR) to the unfolded and folded states of each protein.…”

The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure

“…1B). The fluorescence for both variants decreased slightly after reaching a peak, typical for a protein that aggregates after the melting temperature leading to elimination of available hydrophobic residues that can interact with SYPRO Orange ( 21 ). Since protein aggregation is often concentration dependent, we performed thermal melts at three different protein concentrations.…”

The Immune-Evasive Proline 283 Substitution in Influenza Nucleoprotein Increases Aggregation Propensity Without Altering the Native Structure

“…1B ). The fluorescence for both variants decreased after reaching a peak, likely indicating that NP is aggregating after the melting temperature that eliminates available hydrophobic residues that can interact with SYPRO Orange ( 21 ). Since protein aggregation is often concentration-dependent, we performed thermal melts at two different protein concentrations.…”