| Regional Biophysics Meeting 2005, March 16-20, Zreče, Slovenia | [ProtBiophys] |
There are several proteins that are built up of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of this, no published studies can be found that would address the role of the domain interactions during amyloid formation. A model protein system was developed to investigate the role of domain interactions in the misfolding and amyloid formation of multi-domain proteins. This model 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 microscopy measurements proved that amyloid fibrils could be grown from all mutants under the same conditions as from the wild-type protein. Misfolding and amyloid formation was studied in stopped-flow and manual mixing experiments on the 1 ms to 4 days time-scale. Tryptophan fluorescence was used for selective detection of conformational changes of the domains, accompanying the formation of the amyloidogenic intermediate and the growth of amyloid fibrils. The kinetics of amyloid growth of all the studied mutants could be described using the coagulation model developed for the wild type protein by Modler and coworkers. It occurred that interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, also influencing the final structure. The kinetics of misfolding is different for the individual domains, but similar for the domains within the complete protein. We conclude that the conformational changes of the domains are synchronized by the domain interactions during the misfolding and amyloid formation.
Email: osvath@puskin.sote.hu
Address: Department of Biophysics and Radiation Biology, Semmelweis University, Puskin u. 9, H-1088 Budapest, Hungary