Fast parallel proteolysis (FASTpp)

Fast parallel proteolysis (FASTpp) is a method to determine the thermostability of proteins that does not need previous purification and labelling.

History and background
Proteolysis is widely used in biochemistry and cell biology to probe protein structure. In "limited trypsin proteolysis", low amounts of protease digest both folded and unfolded protein but at largely different rates: unstructured proteins are cut more rapidly, while structured proteins are cut at a slower rate (sometimes by orders of magnitude). Recently, several other assays of protein stability based on proteolysis have been proposed, exploiting other proteases with high specificity for cleaving unfolded proteins. These include Pulse Proteolysis, Proteolytic Scanning Calorimetry and FASTpp.

How it works
FASTpp measures the quantity of protein that resists digestion under various conditions. To this end, a thermostable protease is used, which cleaves specifically at exposed hydrophobic residues. The FASTpp assay combines the thermal unfolding, specificity of a thermostable protease for the unfolded fraction with the separation power of SDS-PAGE. Due to this combination, FASTpp can detect changes in the fraction folded over a large physico-chemical range of conditions including temperatures up to 85°C, pH 6-9, presence or absence of the whole cytosolic proteome. Applications range from biotechnology to study of point mutations and ligand binding assays.

Applications
FASTpp has been used to probe :
 * Lysate effect on protein stability
 * Coupled folding and binding
 * Ligand effects on fraction folded & stability
 * Effects of mutations on fraction folded & stability (e.g. point mutations )
 * Kinetic protein stability

Principles
A protein mixture is aliquoted into several tubes, which are exposed in parallel to different temperatures and a thermostable protease (see figure). Automated temperature control is achieved in a thermal gradient cycler (commonly used for PCRs). Reaction products can be separated by SDS-PAGE or western blot. The protease thermolysin can be fully inactivated by EDTA. This feature of thermolysin makes FASTpp compatible with subsequent trypsin digestion e.g. for mass spectrometry.

Importance of (non)-equilibrium
Many complementary protein stability assays (such as nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy) use (near)equilibrium conditions, which allow for determining free energy differences of unfolding. FASTpp measures in (automatically controlled and therefore highly reproducible) non-equilibrium conditions: It constantly removes the unfolded fraction by cutting at exposed hydrophobic stretches. The proteasome might have a similar effect on unfolded protein in the living eukaryotic cell: Its 20S core can degrade unfolded protein without energy supply.