Abstract 294: Investigating the Small Heat Shock Proteins Impact on Aggregate Inhibition

Abstract

The small heat shock proteins (sHsp) are a highly conserved protein chaperone family needed for ischemia/reperfusion recovery and mitigation of protein aggregation. Without them, environmental stressors, like hypoxia and oxidative stress, can cause highly toxic protein aggregation, leading to cardiomyopathy. Nevertheless, the mechanisms by which sHsps protect cell viability are poorly understood. sHsp mechanism remains elusive due to their highly heterogeneous and dynamic nature making them difficult to study with current methods. To solve this problem, we use a single particle technique called Burst Analysis Spectroscopy (BAS). BAS provides a powerful approach, permitting the real-time observation of size population-resolved kinetics under non-perturbing, free solution conditions. This method allow us to observe events like protein aggregation as well as the interacting sHsp complexes. To study the mechanism of aggregate inhibition by sHsps, we utilize the E. coli sHsps IbpA and IbpB (IbpAB) with two established protein substrates, RuBisCO from R. Rubrum and PepQ from E. coli . Prior work, in vivo and in vitro , has established that IbpAB play a similar role in E. coli as sHsp do in higher eukaryotes suggesting all sHsps may share common mechanistic properties. Using BAS, we established PepQ and RuBisCO form large heterogeneous amorphous aggregates, with RuBisCO also capable of forming fibril-like aggregates under different aggregation conditions. When IbpAB are present during aggregation, both aggregate types decrease in size and form a very specific homogenous aggregate size distribution suggesting IbpAB activity is independent of protein aggregate substrate or structure. Remarkably, binding of IbpAB to RuBisCO aggregates differs dramatically depending on the aggregate structure. The amorphous aggregates appear to bind IbpAB at a RuBisCO monomer-to-IbpAB ratio of 1:1 while the fibril-like RuBisCO aggregates display a bifurcation, where two, distinct aggregate sub-populations bind IbpAB at very different stoichiometries. This data has uncovered the unknown capability of IbpAB/sHsps to inhibit ongoing aggregation of two substrates and structures through two different mechanistic processes all to the same homogenous aggregate size.