The goal of this project is to determine the mechanism of unfolding of substrate proteins during degradation by ATP-dependent proteases. Most protein degradation in cells outside the vacuole or the lysosome is catalyzed by ATP-dependent proteases. Unfolding of the substrate protein is a critical early step in the degradation process. Degradation of short-lived proteins is essential for a wide range of cellular functions including cell cycle control, signal transduction and antigen presentation. Proteolysis of misfolded proteins is part of the cellular response to stress. Barnase, whose folding is well characterized in vitro, was chosen as an artificial substrate protein for the ATP-dependent proteases lon-protease, Clp-protease, mitochondrial AAA-protease and the proteasome. Barnase unfolding during degradation will be compared to spontaneous unfolding to determine whether protein unfolding is catalyzed by the proteases. Destabilized barnase mutants will be used as models for misfolded proteins and barnase mutants with C-terminal degradation tags will be used as models for short-lived regulatory proteins. If unfolding is catalyzed, the mechanism of catalysis will be determined. The educational aim of this award is to teach undergraduate students the prerequisites for a biochemical and biophysical understanding of cell biological processes. For this purpose a laboratory course on quantitative biophysical techniques will be developed.
Lay summary
Proteins are chains made from subunits called amino acids. To perform their biological function, proteins fold into a specific three-dimensional shape. However, there are essential processes in the cell for which proteins must unfold again, such as when damaged or regulatory proteins are degraded by proteases. Failure to degrade regulatory proteins and misfolded proteins leads to abnormal cellular function. This proposal investigates how proteases unfold proteins. Biology has reached a stage where it is possible to determine the detailed mechanisms of processes in the cell. To prepare students for this new era in experimental biology, a laboratory course on quantitative methods in cell biology will be developed.
