Mammalian brain tubulin is an αΒ heterodimer; both α and Β 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 αΒII, αΒIII, and aΒIV 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 Β-tubulin. These compounds have the structure ICH2CONH(CH2)nNHCOCH2I. One of these cross-links, designated Βs, is between cys12 and either cys201 or cys211. The other, designated Β*, is between cys239 and cys354. The Β* cross-link forms in αΒII and αΒIV but not in αΒIII; this is not surprising in view of the fact that αΒIII has serine at position 239 instead of cysteine. However, αΒIII is also unable to form the Βs 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 αΒIII. Although both αΒII and αΒIV can form the Βs cross-link, the dependence on cross-linker chain length is different. αΒII forms Βs with derivatives in which n=2, 4, 5, 6, and 7 but not with those in which n=3 or 10. In contrast, αΒIV forms Βs with derivatives in which n=2, 3, 4, 5, 6, 7, and 10. These results imply that the Βs sulfhydryls are slightly more accessible in αΒIV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that the αΒII, αΒIII, and αΒIV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.
- protein cross-linking
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