Structural characterization and engineering of Adnectin inclusion bodies

Protein aggregation is a fundamental process occurring as a response to aging, disease, protein overexpression and in many areas of biotechnology and medical applications. There has been much recent progress in determining the structural features of protein aggregates that form in cells; yet, owing to prevalent heterogeneity in aggregation, many aspects remain obscure and often experimentally intractable to define. Here we investigate the structural features of unwashed bacterial inclusion bodies (IBs) formed by Adnectins, a family of engineered proteins based on the 10th fibronectin type II domain. These small, stable proteins are actively being designed as biotherapeutics despite being remarkably aggregation-prone, making them an ideal model system for studying aggregation in a biological context. The properties of Adnectin IBs were investigated using a coalition of adapted structural biochemistry techniques including Fourier transform infrared (FTIR) spectroscopy, urea denaturation, Congo red binding and quenched hydrogen deuterium exchange (qHDX) nuclear magnetic resonance (NMR) spectroscopy. It was found that all Adnectin IBs displayed similar structure containing significant native structural features along with a modest contribution from amyloid-like species despite point-mutations affecting their biochemical properties and predicted aggregation behaviour. To further probe aggregate structure, multi- point mutants were rationally designed to investigate how charged residues and monomer stability can influence the ensemble of structures that make up an IB aggregate. As predicted, strongly destabilizing an Adnectin monomer results in an increased contribution from amyloid species. However, these experiments also indicate that charged residues may affect protein stability in ways not anticipated by computational predictors or molecular dynamics simulations, which ultimately demonstrates one of the nuances in understanding the molecular determinants of aggregation as a whole. These findings are of great significance for understanding the specific molecular determinants of IB formation. Specifically, Adnectin variants may more broadly inform on the determinants of aggregation of proteins with immunoglobulin folds, including antibodies which are a rapidly growing group of novel drug candidates