Engineering the Self-Assembly of Bacterial Microcompartment Shell Proteins via Charged Mutations.

Abstract

Protein self-assembly is a fundamental biological process of great importance for the design and synthesis of biomaterials. Developing the ability to precisely manipulate protein assembly would greatly expand both our understanding of the process and our biotechnological capabilities. Within bacteria, proteins that self-organize to form bacterial microcompartments (MCPs) offer an excellent model system for studying protein self-assembly and advancing biomaterial design capabilities. MCPs consist of irregular polyhedral shells that encase an enzyme core that acts as enzymatic nanoreactors. In isolation, the abundant shell proteins of the 1,2-propanediol utilization (Pdu) MCP, PduA and PduJ, have a high propensity to self-assemble into tubular structures, analogous in form to carbon nanotubes. Here, we modulate higher-order assembly of PduA and PduJ hexamers by systematically altering their charge through charge inversion and supercharging across multiple platforms including heterologous overexpression and cell-free protein synthesis. Overexpression and cell-free experiments show that increasing the overall negative charge of assembling subunits consistently promotes self-assembly into tubular structures. Using molecular simulations, we determined the preferred bending angle adopted by the hexameric proteins to predict the most probable self-assembled structures, including honeycomb-like sheets and nanotubes. Simulations of closed PduA and PduJ tubes show the interactions responsible for tube stability, chirality, and radius. <i>In vivo</i>, we find that these charge-altered hexamers are assembly competent within the native MCPs in<i>Salmonella enterica</i>LT2. Our results collectively reveal that both electrostatic interactions and fields generated by charges on proteins can be leveraged to control protein-based nanostructures.