title: Differential dynamics of extracellular and cytoplasmic domains in denatured States of rhodopsin.
abstract: Rhodopsin is a model system for understanding membrane protein folding. Recently, conditions that allow maximally denaturing rhodopsin without causing aggregation have been determined, opening the door to the first structural characterization of denatured states of rhodopsin by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. One-dimensional 1H NMR spectra confirm a progressive increase in flexibility of resonances in rhodopsin with increasing denaturant concentrations. Two-dimensional 1H-15N HSQC spectra of [15N]-α-lysine-labeled rhodopsin in which signals arise primarily from residues in the cytoplasmic (CP) domain and of [15N]-α,ε-tryptophan-labeled rhodopsin in which signals arise only from transmembrane (TM) and extracellular (EC) residues indicate qualitatively that EC and CP domains may be differentially affected by denaturation. To obtain residue-specific information, particular residues in EC and CP domains were investigated by site-directed spin labeling. EPR spectra of the spin-labeled samples indicate that the EC residues retain more rigidity in the denatured states than the CP residues. These results support the notion of residual structure in denatured states of rhodopsin.
authors.affiliation: Department of Structural Biology, University of Pittsburgh School of Medicine , Pittsburgh, Pennsylvania 15260, United States.
authors.forename: Arpana
authors.initials: A
authors.lastname: Dutta
authors.forename: Christian
authors.initials: C
authors.lastname: Altenbach
authors.forename: Sheryll
authors.initials: S
authors.lastname: Mangahas
authors.forename: Naveena
authors.initials: N
authors.lastname: Yanamala
authors.forename: Eric
authors.initials: E
authors.lastname: Gardner
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
authors.forename: Judith
authors.initials: J
authors.lastname: Klein-Seetharaman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry; D002522:Chlorocebus aethiops; D004578:Electron Spin Resonance Spectroscopy; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012243:Rhodopsin / Q000737:chemistry*
chemical_list: D012243:Rhodopsin