Melting proteins in cells

I had a busy Friday, but it was a good day. I attended three different lectures, including one by Rebecca Courtemanche, my MSc student who presented her thesis work. Rebecca has been working on an ongoing project with SGC (Structural Genomics Consortium), in which we are using chemical synthesis in order to develop probes for epigenetic reader proteins. We use a range of methods to tackle this problem (a paper describing some of our advances is now being written in collaboration with the folks at SGC). From my lab’s perspective, this has been an exhilarating experience because my students see first-hand how physicochemical methods such as X-ray crystallography enable them to gain a molecular-level glimpse of how small molecules interact with complex protein targets. There are many methods we use and, while I was listening to Rebecca’s talk, I kept thinking about a paper that really amazed me not too long ago. Dr. Raymond Hui of SGC made me aware of this work. Here is a link to this work:

https://www.sciencemag.org/content/341/6141/84.abstract

A short background is in order: when proteins in their native conformation are denatured, they quickly lose higher-order structures, transitioning to the unfolded states. A thermal shift assay allows you to measure how the melting temperature (Tm, or the temperature at which 50% of the protein is denatured) changes when small molecules interact with proteins. The stronger the binding, the larger is the value of Tm shift (it makes sense, because binding typically stabilizes the folded conformation). This thermal melt assay is one of the central experiments in understanding small molecule/protein binding, especially if your protein has no enzymatic function. The corresponding measurements are typically performed using isolated proteins that are well-behaved and characterized. In the 2013 Science report by Nordlund and colleagues (above), the thermal melt experiment was performed in within cells. This methodology is label free, which is to say there is no need to interfere with the chemical composition of the molecule being investigated, introduce any kinds of linkers, and do anything special for that matter. The method is called cellular thermal shift assay (CETSA). In brief, the authors took aliquots of lysates of mammalian cells, treated them with a drug versus control and heated cells to various temperatures. As the temperature increased, cells were lysed, and denatured protein aggregates (they are insoluble) were separated from their soluble counterparts by centrifugation. Now comes the best part: it was possible to assess the levels of target protein remaining in solution at each temperature by means of Western blotting. If you plot the relative band intensity of the soluble target protein against temperature, the melting temperature of the protein is derived for the free versus the drug-bound. The difference corresponds to the affinity of the interaction. The authors showed that the thermal stability of the target protein was increased when the drug was added in a dose-dependent manner. This is what I call a powerful method.

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