Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

We aim to study the biochemical activity of proteins with unknown functions. Cofactor binding is not only essential for the activity of some proteins, but may also alter their thermal stability. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by the protein's melting temperature, to analyze ligand binding.

Thermal shift assay is a versatile tool for investigating protein interactions with potential cofactors or drugs and for identifying stabilizing conditions for crystallography. Some of its advantages over other screening methods are its relatively simple setup and high throughput with 96 or 384 well plates. Selenoprotein O, or SelO, is an evolutionarily conserved pseudokinase that transfers adenosine monophosphate from ATP to protein substrates in a post-translational modification known as AMPylation In this protocol, we use TSA to analyze nucleotide and metal binding to SelO.

To begin, prepare SelO protein solution using thermal shift assay or TSA buffer. Dispense the protein, small molecules, and SYPRO Orange dye to the appropriate wells of a 384 well plate. Cover the plate with optically clear adhesive film, and briefly spin down the plate at 1000g for one minute in a centrifuge.

Then place the plate in a real-time PCR machine and program it to hold the sample at 20 degrees Celsius for two minutes followed by a 0.5 degree increase in temperature per minute up to 95 degrees Celsius. Export the data from the RTPCR machine for relative fluorescence units, or RFU, with respect to temperature. Using the software of choice, extract and plot data points up to the highest intensity measured for each melting curve.

Determine the melting temperature with the Boltzmann sigmoidal fit for the data to determine the melting temperature, or Tm.The thermal denaturation curves indicated a 4 degrees Celsius increase in the thermal stability of Escherichia coli SelO in the presence of ATP with magnesium ions and a 12 degrees Celsius rise in the same for ATP with manganese ions. However, there was no shift in thermal stability upon incubation with UTP, indicating that Escherichia coli SelO may not exhibit detectable binding to UTP. The no protein as well as the no dye controls had low fluorescence signal which was not responsive to the temperature increase.