title: A time-resolved Förster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein.
abstract: 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 Förster 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.
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98115, USA.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Eustis
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98115, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98115, USA.
authors.forename: Meagan E
authors.initials: ME
authors.lastname: Pilkerton
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98115, USA.
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Whedon
authors.affiliation: Department of Chemistry, University of Washington, Seattle, WA 98115, USA. Electronic address: chatterjee@chem.washington.edu.
authors.forename: Champak
authors.initials: C
authors.lastname: Chatterjee
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D046988:Proteasome Endopeptidase Complex
keywords: Deamidase of Pup; Ligase; Prokaryotic ubiquitin-like protein; Pupylation; TR–FRET; Tuberculosis