(607a) Designing with Nanoscale Building Blocks: Engineering Self-Assembling Protein Superstructures for Applications in Vaccines, Drug Delivery and Biochemical Production

Self-assembling protein containers are promising delivery vehicles for cellular and gene therapy applications, but the ability to predict how mutations alter self-assembly and other particle properties remains a significant challenge. Here, we combine comprehensive codon mutagenesis with high-throughput sequencing to characterize the assembly-competency of all single amino acid variants of several self-assembling proteins, including those from a virus-like particle, a bacterial microcompartment, and a secretion system. The revealed a high-resolution fitness landscape that challenged several conventional protein design assumptions on the composition of linkers, mutability of pore-lining residues, and more. Using the same approach with additional comprehensive mutagenesis strategies and selective pressures on the virus-like particle identified several new variants that permit efficient chemical and post-translational modifications and have altered stability. For example, the wild-type virus-like particle is acid tolerant down to pH 2, but we identified a variant with a single point mutation that confers stability at neutral pH but acid-triggered disassembly. Acid sensitivity is highly desirable in targeted delivery to improve the efficiency of endosomal release. Additional insights will be shared, both from the virus-like particles and from the other two self-assembling model proteins.