| dc.description.abstract | Cells have evolved a set of highly conserved proteins known as chaperones to assist in cellular function and homeostasis by preventing protein misfolding and aggregation. However, under extended stress, some proteins still misfold and aggregate, resulting in diseases such as cardiomyopathies and numerous neuropathies. The small heat shock proteins (sHsps) are a class of chaperones capable of inhibiting aggregation and assisting with protein disaggregation. However, the mechanisms by which the sHsps carry out these functions are poorly understood. In order to address this problem, we have employed the E. coli sHsp, IbpA and IbpB (IbpAB) as a model system, along with an established aggregation-prone substrate RuBisCO (from R. Rubrum). We have found that RuBisCO forms either amorphous or fibril-like aggregates under slightly different conditions. Because standard bulk assays cannot be used to characterize the state distribution of protein aggregates in detail, we have employed a single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS), which permits the minimally perturbative, free-solution observation of aggregate nano-particle size distributions. Using BAS, we observe that IbpAB can limit aggregate particle growth to a strikingly similar but limited size range, which is independent of the aggregation pathway. Using a two-color variant of BAS (MC-BAS), we show that IbpAB displays distinctive binding patterns when interacting with structurally different RuBisCO aggregate particles, suggesting IbpAB can sense and react to specific aggregate characteristics. Using ensemble inter- and intra-molecular FRET assays, we show that the binding of IbpAB to RuBisCO aggregates alters both the average relative proximity of different RuBisCO monomers within an aggregate, as well as the average conformation of the RuBisCO monomer itself. Time-resolved BAS measurements of aggregate disassembly in the presence of the E. coli bi-chaperone disaggregase, consisting of DnaK, DnaJ, GrpE, and ClpB (KJE/B), demonstrate that (1) IbpAB dramatically enhances RuBisCO aggregate disassembly and (2) that IbpAB release from aggregate particles is co-incident with disassembly of the aggregate itself. Additionally, IbpAB can inhibit aggregate growth of an aggregating RuBisCO sample depleted in the non-native monomer pool. However, unlike IbpAB inhibition at the beginning of an aggregating sample, late inhibition does not lead to stimulated disaggregation by the KJE/B disaggregase. Overall, these observations are most consistent with a model in which IbpAB bind to early aggregating states of RuBisCO, incorporate into the nascent aggregate particle and directly alter the conformational properties of the aggregate so that both ongoing particle growth is blocked and subsequent aggregate disassembly is enhanced. | en |
