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Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

Virus-like particles are used to encapsulate drugs, imaging agents, enzymes, and other biologically active molecules in order to enhance their function. However, the size of most virus-like particles is inflexible, precluding the design of appropriately sized containers for different applications. Here, we describe a chromatographic selection for virus-like particle assembly. Using this selection, we identified a single amino acid substitution to the coat protein of bacteriophage MS2 that mediates a uniform switch in particle geometry from T = 3 to T = 1 icosahedral symmetry. The resulting smaller particle retains the ability to be disassembled and reassembled in vitro and to be chemically modified to load cargo into its interior cavity. The pair of 27 and 17 nm MS2 particles will allow direct examination of the effect of size on function in established applications of virus-like particles, including drug delivery and imaging.

Despite abundant genomic and phenotypic data across individuals and environments, the functional impact of most mutations on phenotype remains unclear. Here, we bridge this gap by linking genome to proteome in 800 meiotic progeny from an intercross between two closely related Saccharomyces cerevisiae isolates adapted to distinct niches. Modest genetic distance between the parents generated remarkable proteomic diversity that was amplified in the progeny and captured by 6,476 genotype-protein associations, over 1,600 of which we resolved to single variants. Proteomic adaptation emerged through the combined action of numerous cis - and trans -regulatory mutations, a regulatory architecture that was conserved across the species. Notably, trans -regulatory variants often arose in proteins not traditionally associated with gene regulation, such as enzymes. Moreover, the proteomic consequences of mutations predicted fitness under various stresses. Our study demonstrates that the collective action of natural genetic variants drives dramatic proteome diversification, with molecular consequences that forecast phenotypic outcomes., Competing Interests: Declaration of Interests M. Ralser is founder and shareholder of Eliptica Ltd. The other authors declare no competing interests.