The colchicine-binding and pyrene-excimer-formation activities of tubulin involve a common cysteine residue in the β subunit

Colchicine binding and pyrene excimer fluorescence of tubulin have been used to identify cysteine residue(s) essential for the colchicine binding activity of the protein. We report here that both the colchicine binding activity and the ability to form pyrene excimers of tubulin decay at an identical rate when the protein ages at 37°C. Glycerol, which stabilizes the colchicine binding site also stabilizes the excimer formation equally. Thus, these two properties of tubulin are correlated and are likely to belong to the same structural domain. In an attempt to identify the excimer-forming Cys residues, we found that incubation of tubulin with N,N' ethylenebis (iodoacetamide) causes a significant inhibition of excimer fluorescence. Incubation of tubulin with colchicine prior to this treatment fully retains excimer-forming ability. It is known that Cys239 and Cys354 of β-tubulin, which are about 0.9 nm apart in the native structure, are protected from ethylenebis(iodoacetamide) cross-linking by incubation of tubulin with colchicine [Luduena, R. F. & Roach, M. C. (1981)Pharmacol. Ther. 49, 133-152], These residues must therefore be responsible for the excimer formation of tubulin with pyrene maleimide. Incubation of tubulin with ethylenebis(iodoacetamide) decreases the colchicine binding activity and the excimer formation at an identical rate. Since the alkylation of Cys239 of β-tubulin (responsible for tubulin self-assembly) has no effect on colchicine binding [Bai, R., Lin, C. M., Nguyen, N. Y., Liu, T. & Hamel, E. (1989)Biochemistry 28, 5606-5612], our results suggest that excimer formation and the colchicine binding site of tubulin share Cys354 of the β -subunit. Determination of the number of essential Cys residue(s) for colchicine binding activity, using the statistical method of Tsou [Tsou, C. L. (1962)Sci. Sin. 11, 1535-1558], also shows only one essential Cys residue

Sulfhydryls of tubulin

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 ° C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 ° C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects