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

Abstract

Mammalian brain tubulin is anαΒ heterodimer; bothα andΒ 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 theαΒ _II,αΒ _III, andaΒ _IV tubulin dimers from bovine brain. We have also observed that these isotypes differ in their distributionin vivo and their polymerization and drug-binding propertiesin vitro. We have now explored the question of whether the isotypically purified dimers differ in their overall conformation using as probes compounds of theN,N′-polymethylenebis (iodoacetamide) series which are known to form discrete intrachain cross-links inΒ-tubulin. These compounds have the structure ICH₂CONH(CH₂)_nNHCOCH₂I. One of these cross-links, designatedΒ ^s, is between cys¹² and either cys²⁰¹ or cys²¹¹. The other, designatedΒ ^*, is between cys²³⁹ and cys³⁵⁴. TheΒ ^* cross-link forms inαΒ _II andαΒ _IV but not inαΒ _III; this is not surprising in view of the fact thatαΒ _III has serine at position 239 instead of cysteine. However,αΒ _III is also unable to form theΒ ^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 inαΒ _III. Although bothαΒ _II andαΒ _IV can form theΒ ^s cross-link, the dependence on cross-linker chain length is different.αΒ _II formsΒ ^s with derivatives in whichn=2, 4, 5, 6, and 7 but not with those in whichn=3 or 10. In contrast,αΒ _IV formsΒ ^s with derivatives in whichn=2, 3, 4, 5, 6, 7, and 10. These results imply that theΒ ^s sulfhydryls are slightly more accessible inαΒ _IV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that theαΒ _II,αΒ _III, andαΒ _IV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.