Engineering a Virus-like Particle to Display Peptide Insertions Using an Apparent Fitness Landscape.

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.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification.

Site-specific protein modification is a widely used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically hindered N termini, such as virus-like particles (VLPs) composed of the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.

Grassroots Efforts To Quantify and Improve the Academic Climate of an R1 STEM Department: Using Evidence-Based Discussions To Foster Community.

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.

Experimental Evaluation of Coevolution in a Self-Assembling Particle.

Protein evolution occurs via restricted evolutionary paths that are influenced by both previous and subsequent mutations. This effect, termed epistasis, is critical in population genetics, drug resistance, and immune escape; however, the effect of epistasis on the level of protein fitness is less well characterized. We generated and characterized a 6615-member library of all two-amino acid combinations in a highly mutable loop of a virus-like particle. This particle is a model of protein self-assembly and a promising vehicle for drug delivery and imaging. In addition to characterizing the effect of all double mutants on assembly, thermostability, and acid stability, we observed many instances of epistasis, in which combinations of mutations are either more deleterious or more beneficial than expected. These results were used to generate rules governing the effects of multiple mutations on the self-assembly of the virus-like particle.

Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

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.

Stable Disk Assemblies of a Tobacco Mosaic Virus Mutant as Nanoscale Scaffolds for Applications in Drug Delivery.

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-TMVDOX-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.

A Selection for Assembly Reveals That a Single Amino Acid Mutant of the Bacteriophage MS2 Coat Protein Forms a Smaller Virus-like Particle.

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.

A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein.

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed "undruggable" by conventional methods. Here, we used small-molecule microarray screens to identify and characterize drug-like compounds that modulate the biologic function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacologic ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins.