Subfilamentous protofibril structures in fibrous proteins - Cross-linking evidence for protofibrils in intermediate filaments

The packing of the constituent molecules in some fibrous proteins such as c ollagen and intermediate filaments (IF) is thought to consist of several hi erarchical levels, the penultimate of which is the organization of subfilam entous units termed protofibrils. However, to date only indirect evidence, such as electron microscopic images of unraveling fibers or the existence o f mass quanta, has been adduced in support of the existence of protofibrils . We have reexamined this issue in IF. Cross-links have been induced in tri chocyte keratin, cytokeratin, and vimentin IF proteins. Using improved expe rimental conditions, several additional and reproducible cross-links have b een characterized. Notably, many of these link between columns of molecular strands four apart on two-dimensional surface lattices. These data provide robust support for the concept of an 8-chain (4-molecule) protofibril enti ty in IF. Further, their positions correspond to the axial displacements pr edicted for protofibrils in the different types of IF. Also, the data are c onsistent with intact IF containing four protofibrils. In addition, the pos itions of these novel cross-links suggest that there are multiple possible groupings of four molecular strands to form a protofibril, suggesting a pro miscuous association of molecules to form a protofibril. This may underlie the reason that organized elongated protofibrils cannot be visualized by co nventional microscopic methods.