Reversible denaturation of myo-inositol monophosphatase. The stability of the metal-binding loop.
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.
Moreno
F
F
Unidad de RMN, Universidad Complutense de Madrid, Spain.
Corrales
S
S
Garcia Blanco
F
F
Gore
M G
MG
Rees-Milton
K
K
Churchich
J E
JE
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
D006146:Guanidines; D013725:Terbium; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D019791:Guanidine