title: Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.
abstract: Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain. We report a strong linear correlation between catalytic domain complementation and cyclase activity upon stimulation with forskolin and Gαs. Exploiting this, we dissect the mechanism of action of a series of forskolin analogs and a P-site inhibitor, 2'-d3'-AMP. Finally, we demonstrate that this sensor is functional in live cells, wherein it reports forskolin-stimulated activity of AC.
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota.
authors.forename: Michael
authors.initials: M
authors.lastname: Ritt
authors.affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota sivaraj@umn.edu.
authors.forename: Sivaraj
authors.initials: S
authors.lastname: Sivaramakrishnan
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D000262:Adenylyl Cyclases / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007313:Insecta
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D005576:Colforsin; D000262:Adenylyl Cyclases