FORMATION OF AMYLOID-LIKE FIBRILS BY SELF-ASSOCIATION OF A PARTIALLY UNFOLDED FIBRONECTIN TYPE-III MODULE

The ninth type III module of murine fibronectin was expressed in E. co li and folded into a compact homogeneous monomer whose unfolding and r efolding were then investigated by fluorescence, circular dichroism, c alorimetry and electron microscopy. The isolated module is unusually l abile under physiological conditions. When heated at 1 deg. C/minute i t exhibits an irreversible endothermic transition between 35 and 42 de grees C depending on the protein concentration. The transition is acco mpanied by changes in secondary and tertiary structure with partial ex posure of the single tryptophan and increased binding of the hydrophob ic probe, 1,8-anilinonaphthalene-sulfonate. The partially unfolded int ermediate undergoes rapid self-association leading to the formation of large stable multimers that, like the original monomer, contain subst antial amounts of beta sheet structure. The multimers melt and dissoci ate reversibly in a second endothermic transition between 60 and 90 de grees C also depending on the protein concentration. This second trans ition destroys the remaining secondary structure and further exposes t he tryptophan. Visualization of negatively stained specimens in the el ectron microscope reveals that partially unfolded rmIII-9 slowly forms amyloid-like fibrils of similar to 10 nm width and indeterminate leng th. A subdomain swapping mechanism is proposed in which beta strands f rom one partially unfolded molecule interact with complementary region s of another to form oligomers and polymers. The possibility that simi lar interactions could play a role in the formation of fibrils by fibr onectin in vivo is discussed. (C) 1998 Academic Press.