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

Protein kinases are enzymes that play a key role in cell regulation. One of the best understood kinases from a biochemical point of view is protein kinase A (PKA). PKA is cAMP dependent seronine/threonine kinase, which is composed of two regulatory (PKA-R) and two catalytic (PKA-C) subunits. Investigations on the size and shape of the subunits or the tetrameric holoenzyme is of immense value. Another important parameter is the protein’s melting temperature T_M. Classical techniques to determine hydrodynamic size information include neutron scattering, circular dichroism or analytical gel chromatography. Melting temperatures are commonly measured in thermal shift assays or differential scanning calorimetry. This study demonstrates that the hydrodynamic diameter as well as thermal stability of the catalytic and regulatory PKA subunits could be investigated in the same switchSENSE assay. Changes in the size and shape of proteins could be monitored with two complementary switchSENSE measurement modes (signals): the Dynamic Response measurement mode was used to measure the protein diameter. The Fluorescence Proximity Sensing mode was used for melting experiments. The assay is set up in a few minutes and the screening of molecules which elicit conformational changes or shifts in thermal stability is straight-forward.