title: Equilibrium denaturation studies of mouse beta-nerve growth factor.
abstract: Equilibrium denaturation of dimeric mouse beta-nerve growth factor (beta-NGF) has been studied by monitoring changes in the protein's spectroscopic characteristics. Denaturation of beta-NGF in guanidine hydrochloride and urea resulted in an altered intrinsic fluorescence emission spectrum, fluorescence depolarization, and diminished negative circular dichroism. Native-like spectroscopic properties and specific biological activity are restored when denaturant is diluted from unfolded samples, demonstrating that this process is fully reversible. However, refolding of denatured beta-NGF is dependent on the three disulfide bonds present in the native protein and does not readily occur when the disulfide bonds are reduced. Graphical analysis and nonlinear least-squares fitting of beta-NGF denaturation data demonstrate that denaturation is dependent on the concentration of beta-NGF and is consistent with a two-state model involving native dimer and denatured monomer (N2 = 2D). The conformational stability of mouse beta-NGF calculated according to this model is 19.3 +/- 1.1 kcal/mol in 100 mM sodium phosphate at pH 7. Increasing the hydrogen ion concentration resulted in a 25% decrease in beta-NGF stability at pH 4 relative to pH 7.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: D E
authors.initials: DE
authors.lastname: Timm
authors.forename: K E
authors.initials: KE
authors.lastname: Neet
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
chemical_list: D006146:Guanidines; D009414:Nerve Growth Factors; D014508:Urea; D019791:Guanidine