title: Reversible denaturation of myo-inositol monophosphatase. The stability of the metal-binding loop.
abstract: The unfolding of bovine brain myo-inositol monophosphatase by guanidine. HCl (Gdn. HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn.HCl-treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn.HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal-binding loop. Denaturation of the enzyme by Gdn.HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn.HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1 x 10(6)M-1. It is suggested that a residual structure of denatured myo-inositol monophosphatase is responsible for the binding of terbium ions. The kinetics of unfolding and refolding as a function of Gdn.HCl concentration were monitored by protein fluorescence in a stopped-flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80-65 s-1 at 25 degrees C. The refolding kinetics fit monoexponential functions with rate constants in the range 120-65 s-1 depending on the Gdn.HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.
authors.affiliation: Unidad de RMN, Universidad Complutense de Madrid, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Moreno
authors.forename: S
authors.initials: S
authors.lastname: Corrales
authors.forename: F
authors.initials: F
authors.lastname: Garcia Blanco
authors.forename: M G
authors.initials: MG
authors.lastname: Gore
authors.forename: K
authors.initials: K
authors.lastname: Rees-Milton
authors.forename: J E
authors.initials: JE
authors.lastname: Churchich
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002942:Circular Dichroism; D004795:Enzyme Stability; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008163:Luminescent Measurements; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013725:Terbium / Q000378:metabolism
chemical_list: D006146:Guanidines; D013725:Terbium; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D019791:Guanidine