There are proteins that are built of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single- and multidomain proteins, no published studies can be found that address the role of the domain-domain interactions during misfolding and amyloid formation. By the discovery of the role of domain-domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild-type yeast phosphoglycerate kinase and single-tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild-type protein. Misfolding and amyloid formation was followed in stopped-flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain-domain interactions direct the misfolding and amyloid formation mechanism.
2006 Wiley-Liss, Inc.