CHARACTERIZATION OF DISULFIDE CROSS-LINK FORMATION OF HUMAN VIMENTIN AT THE DIMER, TETRAMER, AND INTERMEDIATE FILAMENT LEVELS

We have investigated the structural interactions of individual molecul es of human vimentin in the soluble state and in filaments. Oxidative crosslinking experiments were conducted with wild-type vimentin aimed at the single cysteine in the helical domain coil 2b, mutated cysteine -free vimentin, and derivatives engineered to carry cysteines in presu med d positions of the heptad amino acid repeats in coils 1a and 2b. W e provide conclusive evidence that crosslinking of the cysteine 328 in wild-type vimentin, when in the filamentous or tetrameric forms, occu rs outside of the coiled-coil dimer, i.e., between staggered dimer mol ecules. This occurs despite the close axial register of the dimers and contradicts previous deductions. The extent of crosslinking increases with temperature as well as with the concentration of the crosslinkin g reagent. We conclude therefore that the cysteines are not in an idea l position for crosslinking but that molecular motion is needed to enh ance the reaction. The occurrence of collision complexes, which has be en speculated in the literature, does not occur and cannot explain the se results. Furthermore, using tailless vimentin with the correspondin g mutations, we provide compelling evidence that in type III intermedi ate filament proteins exchange of individual chains between dimers occ urs only if the proteins are incubated in urea at concentrations above 3 M. In 5 M urea, however, the exchange is completed within seconds. The same reaction occurs between human vimentin and mouse desmin at a comparable speed, indicating that both type III intermediate filament proteins have a high affinity for one another at the coiled-coil level . (C) 1996 Academic Press, Inc.