Development of Fluorescence assays for Studying Protein Disaggregation by Molecular Chaperones

Abstract

The concentrated and complex interior of a cell presents a difficult challenge to folding of many essential proteins. Partially structured folding intermediates, populated during biosynthesis or upon environmental stress, are prone to assemble into large, non-functional aggregates. A network of specialized molecular chaperones evolved to deal with this problem. How aggregate disassembly is accomplished, its impact on disease progression, and how disaggregation is coupled to productive folding is not well understood. We have therefore initiated an effort to establish the enzyme RuBisCo as a model substrate for studying GroEL-dependent protein folding. We have engineered the sequence of RuBisCo so that exogenous fluorescent probes can be coupled to designed surface cysteine residues in a highly specific manner. We are applying fluorescence based techniques to the study of RuBisCO aggregate formation and disassembly by the DnaK-ClpB bi-chaperone system. Our preliminary studies with acid and urea denatured RuBisCo have shown that this protein populates at least two general aggregate assembly pathways, distinguishable by their distinctly different types of aggregate growth. We have also found that the DnaK system (DnaK/DnaJ/GrpE) is capable of altering the progression of RuBisCo aggregation on its own, without either fully arresting aggregation or fully disassembling RuBisCo aggregates. using both light scattering and fluorescence resonance energy transfer (FRET), we find that RuBisCo aggregates are good substrates for the full DnaK-ClpB system.