Peptide insertions in the primary sequence of proteins expand functionality by introducing new binding sequences, chemical handles, or membrane disrupting motifs. With these properties, proteins can be engineered as scaffolds for vaccines or targeted drug delivery vehicles. Virus-like particles (VLPs) are promising platforms for these applications since they are genetically simple, mimic viral structure for cell uptake, and can deliver multiple copies of a therapeutic agent to a given cell. Peptide insertions in the coat protein of VLPs can increase VLP uptake in cells by increasing cell binding, but it is difficult to predict how an insertion affects monomer folding and higher order assembly. To this end, we have engineered the MS2 VLP using a high-throughput technique, called Systematic Mutagenesis and Assembled Particle Selection (SyMAPS). In this work, we applied SyMAPS to investigate a highly mutable loop in the MS2 coat protein to display 9,261 non-native tripeptide insertions. This library generates a discrete map of three amino acid insertions permitted at this location, validates the FG loop as a valuable position for peptide insertion, and illuminates how properties such as charge, flexibility, and hydrogen bonding can interact to preserve or disrupt capsid assembly. Taken together, the results highlight the potential to engineer VLPs in a systematic manner, paving the way to exploring the applications of peptide insertions in biomedically relevant settings.

Women and some racial and ethnic groups remain underrepresented in chemistry departments across the United States, and generally, efforts to improve representation have resulted in minimal or no improvements in the last 10 years. Here, we present the outcomes of a graduate-student-led initiative that sought to assess the issues affecting inclusivity, diversity, and wellness within the Department of Chemistry at the University of California, Berkeley. We report how the results of a department-tailored academic climate survey were used to develop a method to foster open, productive discussion among graduate students, postdoctoral researchers, and faculty. This event format led to an improved understanding of the challenges facing our community members, as well as the identification of strategies that can be used to make the Department of Chemistry more welcoming for all members. We report the success of this student-led effort to highlight the value of assessing diversity and inclusion at the department-level, as well as the benefits of using community data to stimulate productive, evidence-based discussions. Furthermore, we envision that these methods can be implemented within any research-focused academic community to promote positive cultural change.

Current approaches to nanoscale therapeutic delivery rely on the attachment of a drug of interest to a nanomaterial scaffold that is capable of releasing the drug selectively in a tumor environment. One class of nanocarriers receiving significant attention is protein nanomaterials, which are biodegradable and homogeneous in morphology and can be equipped with multiple functional handles for drug attachment. Although most protein-based nanocarriers are spherical in morphology, recent research has revealed that nonspherical nanomaterials may have favorable tumor uptake in comparison to their spherical counterparts. It is therefore important to expand the number of nonspherical protein-based nanocarriers that are available. Herein, we report the development of a self-assembling nanoscale disk derived from a double arginine mutant of recombinantly expressed tobacco mosaic virus coat protein (RR-TMV). RR-TMV disks display highly stable double-disk assembly states. These RR-TMV disks were functionalized with the chemotherapy drug doxorubicin (DOX) and further modified with polyethylene glycol (PEG) for improved solubility. RR-TMV_DOX-PEG displayed cytotoxic properties similar to those of DOX alone when incubated with U87MG glioblastoma cells, but unmodified RR-TMV did not cause any cytotoxicity. The RR-TMV disk assembly represents a promising protein-based nanomaterial for applications in drug delivery.

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.

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.