Pyrene excimer fluorescence as a proximity probe for investigation of residual structure in the unfolded state of human carbonic anhydrase II

FEBS Letters

Hammarström, Per¹  Carlsson, Uno¹  Jonsson, Bengt-Harald²  Kalman, Björn¹ 

[1] IFM-Department of Chemistry, Linköping University, S-581 83 Linköping, Sweden;Department of Biochemistry, Umeå University, S-901 87 Umeå, Sweden

关键词: Excimer fluorescence;    Pyrene;    Folding;    Carbonic anhydrase;    Residual structure;    DMSO;    dimethylsulfoxide;    GuHCl;    guanidine hydrochloride;    HCAII;    human carbonic anhydrase II;    PMIA;    N-(1-pyrenemethyl)iodoacetamide;    py-N67C/C206-py;    pyrene-labeled cysteines in a HCAII mutant with a Asn-67→Cys-67 substitution;   

DOI  :  10.1016/S0014-5793(97)01488-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 β-strand 3, N67C, together with the single naturally occurring cysteine 206 in β-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 β-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.

【 授权许可】

Unknown