Domain interactions direct misfolding and amyloid formation of yeast phosphoglycerate kinase

There are proteins that are built of two structural domains and are deposited full- length in amyloid plaques formed in various dis- eases. 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 interac- tions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate ki- nase 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. Elec- tron microscopic measurements proved that amy- loid 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 syn- chronized indicating that domain- domain interac- tions direct the misfolding and amyloid formation mechanism. Proteins 2006;62:909 -917.