Abstract
Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.
| Original language | English |
|---|---|
| Pages (from-to) | 615-627 |
| Number of pages | 14 |
| Journal | Chem |
| Volume | 10 |
| Issue number | 2 |
| Early online date | 2 Nov 2023 |
| DOIs | |
| Publication status | Published - Feb 2024 |
Bibliographical note
Funding Information:We thank David J. Hamilton for support with compound analytics. We thank the user support team, as well as the beamline staff of I04 at the Diamond Light Source (Oxfordshire, UK) for their support. Funding was provided by the European Research Council ERC proof-of-concept, no. 839088 (T.N.G.) and EU Commission in the framework of the Horizon Europe – EIC Transition Open programme, no. 101057978 (Incircular B.V. S.N.). S.N. S.H. and T.N.G. conceived and designed the project. P.P. and M.I. performed the chemical synthesis. Protein expression and cross-linking was performed by G.H.H. S.K. P.P. I.D. and S.N. MS characterization data processing was performed by A.H.L. R.S. and A.M.R. Enzyme activity measurements, as well as DSF and CD analysis, were performed by G.H.H. S.K. and J.O. Protein crystallography and data processing was performed by S.K. N.M.P. and S.H. All authors analyzed the results. All authors discussed the results and commented on the manuscript. G.H.H. S.N. S.H. and T.N.G. prepared the manuscript. G.H.H. S.K. P.P. I.D. S.N. S.H. and T.N.G. are listed as inventors on a patent application related to the cross-linking of protein complexes. S.N. S.H. and T.N.G. are co-founders and shareholders of Incircular BV, commercializing the corresponding bioconjugation technology. S.H. and T.N.G. are advisers of Incircular BV. We support inclusive, diverse, and equitable conduct of research.
Funding Information:
We thank David J. Hamilton for support with compound analytics. We thank the user support team, as well as the beamline staff of I04 at the Diamond Light Source (Oxfordshire, UK) for their support. Funding was provided by the European Research Council ERC proof-of-concept, no. 839088 (T.N.G.) and EU Commission in the framework of the Horizon Europe – EIC Transition Open programme , no. 101057978 (Incircular B.V., S.N.).
Publisher Copyright:
© 2023 The Author(s)
Funding
We thank David J. Hamilton for support with compound analytics. We thank the user support team, as well as the beamline staff of I04 at the Diamond Light Source (Oxfordshire, UK) for their support. Funding was provided by the European Research Council ERC proof-of-concept, no. 839088 (T.N.G.) and EU Commission in the framework of the Horizon Europe – EIC Transition Open programme, no. 101057978 (Incircular B.V. S.N.). S.N. S.H. and T.N.G. conceived and designed the project. P.P. and M.I. performed the chemical synthesis. Protein expression and cross-linking was performed by G.H.H. S.K. P.P. I.D. and S.N. MS characterization data processing was performed by A.H.L. R.S. and A.M.R. Enzyme activity measurements, as well as DSF and CD analysis, were performed by G.H.H. S.K. and J.O. Protein crystallography and data processing was performed by S.K. N.M.P. and S.H. All authors analyzed the results. All authors discussed the results and commented on the manuscript. G.H.H. S.N. S.H. and T.N.G. prepared the manuscript. G.H.H. S.K. P.P. I.D. S.N. S.H. and T.N.G. are listed as inventors on a patent application related to the cross-linking of protein complexes. S.N. S.H. and T.N.G. are co-founders and shareholders of Incircular BV, commercializing the corresponding bioconjugation technology. S.H. and T.N.G. are advisers of Incircular BV. We support inclusive, diverse, and equitable conduct of research. We thank David J. Hamilton for support with compound analytics. We thank the user support team, as well as the beamline staff of I04 at the Diamond Light Source (Oxfordshire, UK) for their support. Funding was provided by the European Research Council ERC proof-of-concept, no. 839088 (T.N.G.) and EU Commission in the framework of the Horizon Europe – EIC Transition Open programme , no. 101057978 (Incircular B.V., S.N.).
| Funders | Funder number |
|---|---|
| Strategiske Forskningsråd | |
| European Commission | 101057978 |
| European Commission | |
| European Research Council | 839088 |
| European Research Council |
Keywords
- Aggregation
- biocatalysis
- bioconjugation
- chemical biology
- cross-linking
- crystallography
- enzymes
- INCYPRO
- protein engineering
- SDG12: Responsible consumption and production
- SDG9: Industry, innovation, and infrastructure
- structural stabilization
