eprintid: 10196524 rev_number: 6 eprint_status: archive userid: 699 dir: disk0/10/19/65/24 datestamp: 2024-09-04 06:36:43 lastmod: 2024-09-04 06:36:43 status_changed: 2024-09-04 06:36:43 type: article metadata_visibility: show sword_depositor: 699 creators_name: Van de Steen, Alexander creators_name: Wilkinson, Henry C creators_name: Dalby, Paul A creators_name: Frank, Stefanie title: Encapsulation of Transketolase into In Vitro-Assembled Protein Nanocompartments Improves Thermal Stability ispublished: pub divisions: UCL divisions: B04 divisions: F47 keywords: encapsulin, nanocompartment, in vitro assembly, stability, protein, transketolase, cargo loading note: Copyright © 2024 The Authors. Published by American Chemical Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract: Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization. date: 2024-06-04 date_type: published publisher: AMER CHEMICAL SOC official_url: http://dx.doi.org/10.1021/acsabm.3c01153 oa_status: green full_text_type: pub language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 2285382 doi: 10.1021/acsabm.3c01153 medium: Print-Electronic lyricists_name: Frank, Stefanie lyricists_name: Dalby, Paul lyricists_id: SFRAN44 lyricists_id: PADAL59 actors_name: Frank, Stefanie actors_id: SFRAN44 actors_role: owner funding_acknowledgements: EP/R513143/1 [Engineering and Physical Sciences Research Council]; EP/R013756/1 [Engineering and Physical Sciences Research Council]; EP/L015218/1 [Engineering and Physical Sciences Research Council]; [Engineering and Physical Sciences Research Council (EPSRC)]; [Centre for Doctoral Training in Emergent Macromolecular Therapies] full_text_status: public publication: ACS Applied Biomaterials volume: 7 number: 6 pagerange: 3660-3674 pages: 15 event_location: United States issn: 2576-6422 citation: Van de Steen, Alexander; Wilkinson, Henry C; Dalby, Paul A; Frank, Stefanie; (2024) Encapsulation of Transketolase into In Vitro-Assembled Protein Nanocompartments Improves Thermal Stability. ACS Applied Biomaterials , 7 (6) pp. 3660-3674. 10.1021/acsabm.3c01153 <https://doi.org/10.1021/acsabm.3c01153>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10196524/1/Encapsulation%20of%20Transketolase%20into%20iIn%20Vitroi-Assembled%20Protein%20Nanocompartments%20Improves%20Thermal%20Stability.pdf