Excimer fluorescence of pyrene-maleimide-labeled tubulin

Abstract

Excimer-forming cysteines in tubulin are detected by the presence of excimer fluorescence in N-(1-pyrenyl)maleimide-labeled tubulin. The ratio of excimer/monomer fluorescence of labeled protein remained unchanged upon its dilution. These results indicating that both partner of each pair(s) of cysteine are located in the same subunit. The excimer fluorescence is insensitive to prior treatment of tubulin with either colchicine or GTP, indicating that pairs of cysteines protected by those drugs are not involved in excimer formation. This excimer fluorescence of N-(1-pyrenyl)maleimide-labeled tubulin disappeared upon treatment with SDS, guanidinium chloride (GdmCl) and urea. Studies with GdmCl induced unfolding of N-(1-pyrenyl)maleimide-labeled tubulin showed that the loss of excimer fluorescence preceeds subunit dissociation. The loss of both colchicine-binding activity and the excimer fluorescence with increasing temperature indicates a major conformational change of the tubulin molecule at elevated temperatures.