Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry

Abstract

Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding.

EMBER multidimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains

“…The method should be applicable to a variety of amyloids. In principle, it may also be useful to examine dyes that are responsive to molten globule-like intermediates and oligomers ( 59 ). Moreover, EMBER may be useful to rapidly screen hundreds of neurodegenerative disease donor samples and prioritize new targets for cryo-EM structural characterization.…”

EMBER multi-dimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains

“…In principle, it may also be useful for examine dyes that are responsive to molten globule-like intermediates and oligomers. 59 Moreover, EMBER may be useful to rapidly screen hundreds of neurodegenerative disease donor samples and prioritize new targets for cryo-EM structural characterization.…”

Navigating the Expansive Landscapes of Soft Materials: A User Guide for High-Throughput Workflows

“…While DSC is limited in throughput, differential scanning fluorimetry (DSF) can rapidly detect thermal transitions by tracking changes in solvatochromic fluorescent dyes with affinity for hydrophobic regions of a protein that become exposed upon unfolding . DSF can also probe binding interactions between proteins and small molecules or polymer networks ,, with low sample burden (10 pmol) by measuring temperature-dependent fluorescence changes with simultaneous real time-PCR instrumentation . However, DSF depends strongly on protein-reporter interactions and not every reporter will capture relevant thermal shifts …”

“…Dyes not coupled to the polymer can span solvatochromic, colorimetric, or fluorogenic types, and are capable of characterizing diverse properties including conformational changes, , aggregation, , cell viability, , metal ion uptake, ,− and catalytic activity. , Characterization using dyes is both accessible and straightforward, involving minimal sample preparation and rapid throughput in well plates. For example, Nile red is a fluorescent dye used to visualize hydrophobic surfaces of proteins, polymers, and lipid droplets .…”

“…5 This technique also requires a fluorescent probe, and has been used to determine the fluidity or rigidity of lipid and polymer membranes 235 and yield time-resolved conformation and dynamics analysis of polymers in solution. 236−238 Dyes not coupled to the polymer can span solvatochromic, colorimetric, or fluorogenic types, and are capable of characterizing diverse properties including conformational changes, 98,214 aggregation, 239,240 cell viability, 241,242 metal ion uptake, 105,243−245 and catalytic activity. 45,46 Characterization using dyes is both accessible and straightforward, involving minimal sample preparation and rapid throughput in well plates.…”