Protein-adaptive differential scanning fluorimetry using conformationally responsive dyes.

Differential scanning fluorimetry (DSF) is a technique that reports protein thermal stability via the selective recognition of unfolded states by fluorogenic dyes. However, DSF applications remain limited by protein incompatibilities with existing DSF dyes. Here we overcome this obstacle with the development of a protein-adaptive DSF platform (paDSF) that combines a dye library 'Aurora' with a streamlined procedure to identify protein-dye pairs on demand. paDSF was successfully applied to 94% (66 of 70) of proteins, tripling the previous compatibility and delivering assays for 66 functionally and biochemically diverse proteins, including 10 from severe acute respiratory syndrome coronavirus 2. We find that paDSF can be used to monitor biological processes that were previously inaccessible, demonstrated for the interdomain allostery of O-GlcNAc transferase. The chemical diversity and varied selectivities of Aurora dyes suggest that paDSF functionality may be readily extended. paDSF is a generalizable tool to interrogate protein stability, dynamics and ligand binding.

Protocol for performing and optimizing differential scanning fluorimetry experiments.

Differential scanning fluorimetry (DSF) is a widely used technique for determining the apparent melting temperature (Tma) of a purified protein. Here, we present a protocol for performing and optimizing DSF experiments. We describe steps for designing and performing the experiment, analyzing data, and optimization. We provide benchmarks for typical Tmas and ΔTmas, standard assay conditions, and upper and lower limits of commonly altered experimental variables. We also detail common pitfalls of DSF and ways to avoid, identify, and overcome them.

Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry.

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.

Roundup negatively impacts the behavior and nerve function of the Madagascar hissing cockroach (Gromphadorhina portentosa).

Glyphosate is the active ingredient in Roundup formulations. Glyphosate-based herbicides are used globally in agriculture, forestry, horticulture, and in urban settings. Glyphosate can persist for years in our soil, potentially impacting the soil-dwelling arthropods that are primary drivers of a suite of ecosystem services. Furthermore, although glyphosate is not generally classified as neurotoxic to insects, evidence suggests that it may cause nerve damage in other organisms. In a series of experiments, we used food to deliver environmentally realistic amounts of Roundup ready-to-use III, a common 2% glyphosate-based herbicide formulation that lists isopropylamine salt as its active ingredient, to Madagascar hissing cockroaches. We then assessed the impact of contamination on body mass, nerve health, and behavior. Contaminated food contained both 30.6 mg glyphosate and so-called inert ingredients. Food was refreshed weekly for 26-60 days, depending on the experiment. We found that consumption of contaminated food did not impact adult and juvenile survivorship or body weight. However, consumption of contaminated food decreased ventral nerve cord action-potential velocity by 32%, caused a 29% increase in respiration rate, and caused a 74.4% decrease in time spent on a motorized exercise wheel. Such changes in behavior may make cockroaches less capable of fulfilling their ecological service, such as pollinating or decomposing litter. Furthermore, their lack of coordination may make them more susceptible to predation, putting their population at risk. Given the decline of terrestrial insect abundance, understanding common risks to terrestrial insect populations has never been more critical. Results from our experiments add to the growing body of literature suggesting that this popular herbicide can act as a neurotoxin.