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1.
ACS Synth Biol ; 13(1): 157-167, 2024 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-38133598

RESUMEN

Protein nanocages have diverse applications in medicine and biotechnology, including molecular delivery. However, although numerous studies have demonstrated the ability of protein nanocages to encapsulate various molecular species, limited methods are available for subsequently opening a nanocage for cargo release under specific conditions. A modular platform with a specific protein-target-based mechanism of nanocage opening is notably lacking. To address this important technology gap, we present a new class of designed protein cages, the Ligand-Operable Cage (LOC). LOCs primarily comprise a protein nanocage core and a fused surface binding adaptor. The geometry of the LOC is designed so that binding of a target protein ligand (or multiple copies thereof) to the surface binder is sterically incompatible with retention of the assembled state of the cage. Therefore, the tight binding of a target ligand drives cage disassembly by mass action, subsequently exposing the encapsulated cargo. LOCs are modular; direct substitution of the surface binder sequence can reprogram the nanocage to open in response to any target protein ligand of interest. We demonstrate these design principles using both a natural and a designed protein cage as the core, with different proteins acting as the triggering ligand and with different reporter readouts─fluorescence unquenching and luminescence─for cage disassembly. These developments advance the critical problem of targeted molecular delivery and detection.


Asunto(s)
Biotecnología , Proteínas , Unión Proteica , Ligandos , Proteínas/química , Fluorescencia
2.
Protein Sci ; 33(4): e4973, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38533546

RESUMEN

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, for example, so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.


Asunto(s)
Proteínas de Repetición de Anquirina Diseñadas , Proteínas , Proteínas/química
3.
bioRxiv ; 2023 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-37986890

RESUMEN

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, e.g., so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

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