A time-resolved Forster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein

The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup, called Dop, has no known mammalian homologs. Because Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for tuberculosis therapy. To facilitate high-throughput screens for Dop inhibitors, we developed a time-resolved Forster resonance energy transfer (TR-FRET)-based assay for Dop function. The TR FRET assay was successfully applied to determine the Michaelis constant for adenosine triphosphate (ATP) binding and to test the cofactor tolerance of Dop. Published by Elsevier Inc.