title: Binding of the volatile general anesthetics halothane and isoflurane to a mammalian beta-barrel protein.
abstract: A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic-protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large beta-barrel internal cavity (515 +/- 30 angstroms(3)) lined predominantly by aromatic and aliphatic residues. Halothane binding to the beta-barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the beta-barrel of porcine odorant binding protein with dissociation constants of 0.46 +/- 0.10 mM and 0.43 +/- 0.12 mM determined using fluorescence quenching and competitive binding with 1-aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with K(d) values of 80 +/- 10 microM and 100 +/- 10 microM, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by -0.9 +/- 0.1 kcal.mol(-1). In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X-ray diffraction to 2.25 angstroms resolution [1], this represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules.
authors.affiliation: Department of Anesthesia, University of Pennsylvania, Philadelphia, PA 19104, USA. JohanssJ@uphs.upenn.edu
authors.forename: Jonas S
authors.initials: JS
authors.lastname: Johansson
authors.forename: Gavin A
authors.initials: GA
authors.lastname: Manderson
authors.forename: Roberto
authors.initials: R
authors.lastname: Ramoni
authors.forename: Stefano
authors.initials: S
authors.lastname: Grolli
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
mesh_terms: D018685:Anesthetics, Inhalation / Q000378:metabolism*; D000818:Animals; D002151:Calorimetry; D006221:Halothane / Q000378:metabolism*; D007530:Isoflurane / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D018035:Receptors, Odorant / Q000737:chemistry* / Q000378:metabolism; D013552:Swine; D014961:X-Ray Diffraction
chemical_list: D018685:Anesthetics, Inhalation; D018035:Receptors, Odorant; C045648:odorant-binding protein; D007530:Isoflurane; D006221:Halothane