Experimental Evaluation of Coevolution in a Self-Assembling Particle

Hartman, Emily C.; Lobba, Marco; Favor, Andrew H.; Robinson, Stephanie A.; Francis, Matthew B.; Tullman‐Ercek, Danielle

doi:10.1021/acs.biochem.8b00948

Cited by 22 publications

(41 citation statements)

References 68 publications

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“…[20][21][22] Limited genetic manipulations can be made to the MS2 coat protein (CP) without disrupting the assembly state, 23 and many interand intra-subunit contacts make mutability challenging to predict. 24 Additionally, the native N terminus is sterically hindered, and efforts to extend the N terminus have had limited success. 25 As such, currently developed N-terminal modification strategies are not compatible with the MS2 CP.…”

mentioning

confidence: 99%

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

Brauer,

Hartman,

Bader

et al. 2019

J. Am. Chem. Soc.

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Site-Specific Modification. ChemRxiv. Preprint. 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 like 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. File list (3) download file view on ChemRxiv ChemRxiv Submission Brauer Hartman.pdf (2.67 MiB) download file view on ChemRxiv Supplementary Information Brauer Hartman.pdf (2.78 MiB) download file view on ChemRxiv Supplementary Data-Primers.xlsx (16.69 KiB)

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mentioning

confidence: 99%

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

Brauer,

Hartman,

Bader

et al. 2019

J. Am. Chem. Soc.

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“…These constraints can indicate where modifications are likely to succeed, although not what form they should take. We will see that in MS2, the gauge points reveal critical structural locations which are nearly impossible to modify [23,24].…”

Section: Resultsmentioning

confidence: 84%

“…Equivalent capsid proteins are colored blue, gray and cyan. Though this fit uses only 2 of the 5 radial levels of the point arrays, its agreement with experiments is remarkable, as it is nearly impossible to substitute different amino acids near the gauge points (orange) [23,24]. (b) A perspective view of the gauge points sitting right next to the pentamer protrusions, which were found to be nearly immutable, suggesting this geometric location is critical to the stability of MS2.…”

Section: Hepatitis B (Hbv T = 4 1qgt)mentioning

confidence: 76%

“…Knowledge of the interior structure of the ssRNA should enable us to distinguish between these two point arrays. Recently a series of very clever experiments were conducted by Hartman et al [23,24], where they systemically replaced every amino acid, one by one, of the subunit proteins of MS2 with each of the 19 other possible α-amino acids. As MS2 encapsulates its RNA genome upon formation of the capsid, they were able to determine which locations they could swap out and still form a stable virus.…”

Section: Bacteriophage Ms2 (Ms2 T = 3 2ms2)mentioning

confidence: 99%

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Unveiling the Hidden Rules of Spherical Viruses Using Point Arrays

Wilson

2020

Viruses

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Since its introduction, the Triangulation number has been the most successful and ubiquitous scheme for classifying spherical viruses. However, despite its many successes, it fails to describe the relative angular orientations of proteins, as well as their radial mass distribution within the capsid. It also fails to provide any critical insight into sites of stability, modifications or possible mutations. We show how classifying spherical viruses using icosahedral point arrays, introduced by Keef and Twarock, unveils new geometric rules and constraints for understanding virus stability and key locations for exterior and interior modifications. We present a modified fitness measure which classifies viruses in an unambiguous and rigorous manner, irrespective of local surface chemistry, steric hinderance, solvent accessibility or Triangulation number. We then use these point arrays to explain the immutable surface loops of bacteriophage MS2, the relative reactivity of surface lysine residues in CPMV and the non-quasi-equivalent flexibility of the HBV dimers. We then explain how point arrays can be used as a predictive tool for site-directed modifications of capsids. This success builds on our previous work showing that viruses place their protruding features along the great circles of the asymmetric unit, demonstrating that viruses indeed adhere to these geometric constraints.

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“…We applied this technique to explore the mutability and principles of higher order assembly for the MS2 VLP. [17][18][19] Through this work we identified a region known as the "FG loop" to be especially permissive to mutations. This loop is unique in its conformations and placement in the packing of MS2 monomers.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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 . CC-BY-NC-ND 4.

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Experimental Evaluation of Coevolution in a Self-Assembling Particle

Cited by 22 publications

References 68 publications

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

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

Unveiling the Hidden Rules of Spherical Viruses Using Point Arrays

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

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