An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I
MS) method for ...charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.
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.
Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial ...strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle’s assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle’s mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS’s assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.
Virus-like particles (VLPs) are self-assembling protein nanoparticles that have great promise as vectors for drug delivery. VLPs are derived from viruses but retain none of their infection or ...replication capabilities. These protein particles have defined surface chemistries, uniform sizes, and stability properties that make them attractive starting points for drug-delivery scaffolds. Here, we review recent advances in tailoring VLPs for drug-delivery applications, including VLP platform engineering approaches as well as methods for cargo loading, activation, and release. Finally, we highlight several successes using VLPs for drug delivery in model systems.
•Virus-like particles (VLPs) are versatile structuresfor drug-delivery applications.•Mutations to VLP structural proteins can improve VLP drug delivery.•VLP–cargo loading, release, and activation are key traits for drug delivery.
Virus-like particles (VLPs) are promising scaffolds for biomaterials as well as diagnostic and therapeutic applications. However, there are some key challenges to be solved, such as the ability to ...engineer alternate sizes for varied use cases. To this end, we created a library of MS2 VLP variants at two key residues in the coat protein which have been implicated as important to controlling VLP size and geometry. By adapting a method for systematic mutagenesis coupled with size-based selections and high-throughput sequencing as a readout, we developed a quantitative assessment of two residues in MS2 coat protein that govern the size shift in MS2 VLPs. We then applied the strategy to the equivalent residues in Qβ VLPs, an MS2 homolog, and demonstrate that the analogous pair of residues are also able to impact Qβ VLP size and shape. These results underscore the power of fitness landscapes in identifying critical features for assembly.
•Development of a high throughout selection to quantitatively assess particle size.•Confirmation of a critical residue in the formation of smaller sized MS2 VLPs.•Extrapolation of method and critical residue data to create smaller Qβ-based VLPs.•Mutations to unstructured loop regions influence presence of mini-sized VLPs.
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