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Stability

What is the problem? Biophysical assays can be difficult to complete due to the inherently unstable nature of proteins when they are isolated and over concentrated, resulting in aggregation, oxidation and denaturation.

How do we deal with it? Short of changing the construct, protein stability can be increased by optimizing the formulation (formulation: a mixture of two or more chemicals) that the protein is stored in.  The challenge is to find which of the numerous formulation options available is best for any given protein, and we think that thermal stability as measured by differential scanning fluorimetry (DSF) is the most appropriate way to do so.

We’ve composed a trilogy of papers about DSF

Paper 1) Describing Buffer Screen 9, which is our current approach: Seabrook & Newman, ACS Combinatorial Science

Paper 2) The development of Meltdown to automate analysis: Rosa et al., Journal of Biomolecular Screening

Paper 3) A statistical reflection on the usefulness of the assay: Ristic et al., Acta Crystallography Section F

Protein unfolding and dye binding event

How does it work? DSF works by using a dye (SYPRO) that is fluorescent active when bound to a hydrophobic environment, i.e. the core of an unfolded protein – see the image above. Our implementation of DSF works using a fairly standard RT-PCR machine that allows appropriate excitation and emission wavelengths for active SYPRO dye. We scan against a suite of formulations based on 15 buffers with both low and high salt concentrations (Buffer Screen 9 – contents available on the C6 webtool), from 20C to 100C.

What is the output? The resulting melt curve looks like the image below. The melt temperature, correctly termed the temperature of hydrophobic exposure, is found as the peak of the first derivative of the melt curve. An increase in melt temperature indicates that more energy was required to denature the protein, which is correlated with conformation stability (which should give a longer life span from a single batch of protein).

Classic DSF melt curve (solid line), first derivative (dashed curve)

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NEXT: Data Analysis, Meltdown