Use of N,N'-polymethylenebis(iodoacetamide) derivatives as probes for the detection of conformational differences in tubulin isotypes.

Abstract

Mammalian brain tubulin is an alpha beta heterodimer; both alpha and beta exist in 6-7 isotypic forms which differ in their amino acid sequences. By the use of isotype-specific monoclonal antibodies, we have previously shown that we can purify the alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain. We have also observed that these isotypes differ in their distribution in vivo and their polymerization and drug-binding properties in vitro. We have now explored the question of whether the isotypically purified dimers differ in their overall conformation using as probes compounds of the N,N'-polymethylenebis (iodoacetamide) series which are known to form discrete intrachain cross-links in beta-tubulin. These compounds have the structure ICH2CONH(CH2)nNHCOCH2I. One of these cross-links, designated beta s, is between cys12 and either cys201 or cys211. The other, designated beta*, is between cys239 and cys354. The beta* cross-link forms in alpha beta II and alpha beta IV but not in alpha beta III; this is not surprising in view of the fact that alpha beta III has serine at position 239 instead of cysteine. However, alpha beta III is also unable to form the beta s cross-link, although it appears to have all three cysteines which may be involved in the cross-link. This suggests that at least one of the sulfhydryls involved in the cross-link may be inaccessible in alpha beta III. Although both alpha beta II and alpha beta IV can form the beta s cross-link, the dependence on cross-linker chain length is different. alpha beta II forms beta s with derivatives in which n = 2, 4, 5, 6, and 7 but not with those in which n = 3 or 10. In contrast, alpha beta IV forms beta s with derivatives in which n = 2, 3, 4, 5, 6, 7, and 10. These results imply that the beta s sulfhydryls are slightly more accessible in alpha beta IV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that the alpha beta II, alpha beta III, and alpha beta IV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.