title: Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II.
abstract: The excimer fluorescence from two pyrenyl moieties attached to cysteines in human carbonic anhydrase II has been monitored to characterize residual structure retained under strong denaturing conditions. A position in beta-strand 3, N67C, together with the single naturally occurring cysteine 206 in beta-strand 7, were used as attachment sites. The eximer formation by the pyrenyls, requiring proximity of the probes, revealed an unfolding transition at a GuHCl concentration significantly higher than that required to induce unfolding of the molten globule state as monitored by CD. These results indicate that the excimer transition monitors the unfolding of a residual compact structure that spans beta-strands 3-7. This region constitutes the central and the most hydrophobic part of the molecule, emphasizing the importance of hydrophobic interaction in maintaining residual structure under strong unfolding conditions.
authors.affiliation: IFM-Department of Chemistry, Linköping University, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Hammarström
authors.forename: B
authors.initials: B
authors.lastname: Kalman
authors.forename: B H
authors.initials: BH
authors.lastname: Jonsson
authors.forename: U
authors.initials: U
authors.lastname: Carlsson
mesh_terms: D002256:Carbonic Anhydrases / Q000737:chemistry*; D002942:Circular Dichroism; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D007460:Iodoacetamide / Q000031:analogs & derivatives*; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D011721:Pyrenes; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; C110226:N-(1-pyrenemethyl)iodoacetamide; D011721:Pyrenes; D002256:Carbonic Anhydrases; D019791:Guanidine; D003545:Cysteine; D007460:Iodoacetamide