Subfilamentous protofibril structures in fibrous proteins: cross-linking evidence for protofibrils in intermediate...

The packing of the constituent molecules in some fibrous proteins such as collagen and intermediate filaments (IF) is thought to consist of several hierarchical levels, the penultimate of which is the organization of subfilamentous units termed protofibrils. However, to date only indirect evidence, such as electron microscopic images of unraveling fibers or the existence of 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 trichocyte keratin, cytokeratin, and vimentin IF proteins. Using improved experimental conditions, several additional and reproducible cross-links have been 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 entity in IF. Further, their positions correspond to the axial displacements predicted for protofibrils in the different types of IF. Also, the data are consistent with intact IF containing four protofibrils. In addition, the positions of these novel cross-links suggest that there are multiple possible groupings of four molecular strands to form a protofibril, suggesting a promiscuous association of molecules to form a protofibril. This may underlie the reason that organized elongated protofibrils cannot be visualized by conventional microscopic methods.