The Impact Of Hidden Aggregate Structure On Molecular Chaperone Disaggregation Revealed By Single Particle Fluorescence Burst Analysis
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Molecular chaperones are tasked with folding and disassembling the misfolded and aggregated non-native proteins that arise during biosynthesis or upon environmental stress. However, the heterogeneous and dynamic nature of misfolded and aggregated non-native proteins makes it challenging to study the mechanism of protein disaggregation by molecular chaperones. A central unresolved question is whether it is the size or structure of non-native protein aggregates that limits how well cells clear potentially toxic aggregates. The work described herein demonstrates that protein aggregate size does not intrinsically limit how well protein aggregates are dismantled by molecular chaperones. Rather, the primary constraint appears to be the internal structure of aggregates. This conclusion is made possible, in part, by the application of a fluorescence-based single particle technique known as Burst Analysis Spectroscopy (BAS). Several novel extensions to the BAS method, that substantially extend both the power and general applicability of the method, are developed as part of this work and are also presented here. The potential of extensions that permit multi-color analysis and extend the analyzable size range are demonstrated in case studies of DNA packing in lambda phage, protein aggregation, and ENTH mediated vesiculation. The CO2-fixing enzyme ribulose-1, 5-bisphosphate-carboxylase oxygenase (RuBisCO) from R. rubrum was employed as the model substrate protein in the study of protein disaggregation by molecular chaperones. Using BAS, non-native RuBisCO is shown to follow at least two general aggregation pathways, which can be distinguished by their unique population distributions and growth behavior. Visualized by EM, RuBisCO aggregates that grow slowly (slow growing) are amorphous in shape, while rapidly growing aggregates (fast growing) are fibroid-like. These two chemically identical but structurally and conformationally distinct aggregates respond very differently to active disaggregation by a model molecular chaperone network consisting of the E. coli Hsp70 and Hsp100 molecular chaperones. While fast growing aggregates can be disassembled by the Hsp70 system alone, slow growing aggregates require both the Hsp70 and Hsp100 systems. In all cases, BAS measurements demonstrate that the efficiency of RuBisCO aggregate disassembly is not impacted by aggregate size. Strikingly, slow growing aggregates display a dramatic shift in behavior with time, becoming increasingly refractory to disassembly with no detectable changes in size, and without losing the ability to bind Hsp70. Using inter-molecular fluorescence resonance energy transfer (FRET) measurements, it is shown that this shift in behavior is consistent with a compaction or tightening of the average aggregate structure. Additionally, the Hsp70 system is shown to alter this structure without inducing disassembly, consistent with models in which Hsp70s play a key role in preparing or loosening aggregates prior to engagement by Hsp100s.
Shoup, Daniel Wayne (2016). The Impact Of Hidden Aggregate Structure On Molecular Chaperone Disaggregation Revealed By Single Particle Fluorescence Burst Analysis. Doctoral dissertation, Texas A & M University. Available electronically from