Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II.
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.
Hammarström
P
P
IFM-Department of Chemistry, Linköping University, Sweden.
Kalman
B
B
Jonsson
B H
BH
Carlsson
U
U
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
D005456:Fluorescent Dyes; C110226:N-(1-pyrenemethyl)iodoacetamide; D011721:Pyrenes; D002256:Carbonic Anhydrases; D019791:Guanidine; D003545:Cysteine; D007460:Iodoacetamide