Hydrodynamic diameter and thermal stability measurements of protein kinase A using switchSENSE®

Protein kinases are enzymes that play a key role in cellular regulation. One of the best understood kinases from a biochemical point of view is protein kinase A (PKA). PKA is a cAMP dependent seronine/threonine kinase, which is composed of two regulatory (PKA-R) and two catalytic (PKA-C) subunits. The enzyme undergoes multiple conformational changes and thus, information on size and shape of the subunits, or the tetrameric holoenzyme is of immense value. Another important parameter which is indicative of a correctly folded protein and if a protein’s binding pocket for small molecule is occupied or vacant, is the protein’s melting temperature TM. Classical techniques to determine hydrodynamic size information include neutron scattering, circular dichroism or analytical gel chromatography. Melting temperatures are commonly measured employing thermal shift assays or differential scanning calorimetry. Here we show for the first time how both parameters, DH and TM, are measured simultaneously in the same assay setup. Methods and Results