title: Detergents stabilize the conformation of phosphodiesterase 6.
abstract: Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the α and β subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDEγ (Pγ) subunits, and binding of two types of isoprenyl-binding protein δ, called PrBP/δ, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/δ and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/δ from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.
authors: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States.
authors: Bo Y
authors: BY
authors: Baker
authors: Krzysztof
authors: K
authors: Palczewski
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000187:drug effects; D002417:Cattle; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents / Q000737:chemistry*; D004795:Enzyme Stability / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D059748:Proteolysis; D013012:Sorbitol / Q000031:analogs & derivatives / Q000737:chemistry; D013395:Sucrose / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: D003902:Detergents; C018152:sucrose monolaurate; D013012:Sorbitol; D013395:Sucrose; C063161:MEGA 8; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6