The transition state structure of protein aggregation

点击翻译按钮获取中文摘要

Proteins are the most versatile and complex class of all known molecules. Most biological processes crucially require one or more proteins in a very specific structural conformation and protein molecules often fold spontaneously into these structures. The mechanisms of protein folding have been studied in the last 20 years at the single amino acid residue level of detail, thanks to the advances of spectroscopic techniques and methodologies such as Phi-value analysis, a combination of accurate kinetic and thermodynamic measurements with protein engineering, allowing determination of the structures of the folding transition states. However, other than fold into their native state, proteins can also aggregate into ordered supramolecular structures, in particular into amyloid fibrils, and such processes are a hallmark of a range of severe neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Our fundamental mechanistic understanding of these aggregation processes lacks far behind that of protein folding. This lack of progress is partly due to the complex multistep nature of the aggregation process, which consists of nucleation, growth and potentially autocatalytic amplification steps, but also due to a lack of suitable experimental strategies to tackle this challenge. In this research project, I intend to develop a comprehensive approach to address this problem. The experimental strategy I propose consists of developing a variant of the Phi-value analysis (which has proven highly successful in the study of protein folding) which can be applied to protein misfolding and aggregation. This is in particular only possible for the growth step of the overall aggregation process. These experiments will be performed with a small protein domain (PI3K-SH3), because extensive data exists on the folding mechanism of SH3 domains, with which the data on the aggregation mechanism of this protein that will be obtained here, can be compared. The aim is hereby to gain structural information on the transition state, the state of highest energy between the monomeric and the aggregated protein. This will be the first experimental strategy to push the limits of mechanistic understanding of protein self-assembly and aggregation down to the level of individual amino acid residues.Furthermore, I aim to develop in this project a novel high throughput experimental strategy, with which it will be possible in the future to investigate a significantly larger number of sequence variant (hundreds, instead of dozens). The long term goal of this research program is to elucidate the exact aggregation mechanism of various proteins, in order to understand better the origin of this general phenomenon and in order to be able to control it.