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In situ cyclization of proteins

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Protein engineering technology

The in situ cyclization of proteins (INCYPRO) is a protein engineering technology that increases the durability of proteins and enzymes for biotechnological and biomedical applications. For such applications, it is essential that the used proteins maintain their structural integrity. This is, however, often challenged due to the conditions required for these applications which necessitates protein engineering to stabilize the protein structure. The INCYPRO technology involves the attachment of molecular claps (crosslinks) to a protein, thereby reducing the tendency of the protein to unfold. The resulting INCYPRO-crosslinked proteins are more stable at elevated temperature and in presence of chemical denaturants.

Technology

The INCYPRO technology utilizes tris-reactive molecules to crosslink three defined positions within a protein or protein complex. For example, INCYPRO can involve the introduction of three spatially aligned and solvent-accessible cysteines into the protein that are then reacted with a tris-electrophilic agent. The resulting crosslinked proteins or protein complexes have been shown to exhibit increased stability towards thermal and chemical stress and a lower tendency towards aggregation. So far, the melting temperature of proteins was increased by up to 39°C in a single design step.

Examples

An early example, involved the stabilization of the transpeptidase Sortase A which resulted in INCYPRO-stabilized variants with activity under elevated temperature and in the presence of guanidinium chloride. INCYPRO has also been applied to stabilize the human adaptor KIX domain utilizing different crosslinker molecules. Here, a dependency of protein stability on the hydrophilicity of the crosslink was observed. In addition, a number of homo-trimeric protein complexes was stabilized including the Pseudomonas fluorescens esterase (PFE) and an Enoyl-CoA hydratase. In these cases, enzyme conjugates with overall bicyclic topology were generated.

See also

References

  1. ^ Pelay-Gimeno M, Bange T, Hennig S, Grossmann TN (August 2018). "In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme". Angewandte Chemie. 57 (35): 11164–11170. doi:10.1002/anie.201804506. PMC 6120448. PMID 29847004.
  2. ^ Neubacher S, Saya JM, Amore A, Grossmann TN (February 2020). "In Situ Cyclization of Proteins (INCYPRO): Cross-Link Derivatization Modulates Protein Stability". The Journal of Organic Chemistry. 85 (3): 1476–1483. doi:10.1021/acs.joc.9b02490. PMC 7011175. PMID 31790232.
  3. Gligorijević V, Renfrew PD, Kosciolek T, Leman JK, Berenberg D, Vatanen T, et al. (May 2021). "Structure-based protein function prediction using graph convolutional networks". Nature Communications. 12 (1): 3168. Bibcode:2021NatCo..12.3168G. doi:10.1038/s41467-021-23303-9. PMC 8155034. PMID 34039967.
  4. Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (May 2012). "Engineering the third wave of biocatalysis". Nature. 485 (7397): 185–194. Bibcode:2012Natur.485..185B. doi:10.1038/nature11117. PMID 22575958. S2CID 4379415.
  5. ^ Kiehstaller S, Hutchins GH, Amore A, Gerber A, Ibrahim M, Hennig S, et al. (June 2023). "Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions". Bioconjugate Chemistry. 34 (6): 1114–1121. doi:10.1021/acs.bioconjchem.3c00151. PMC 10288436. PMID 37246906.
  6. ^ Hutchins GH, Kiehstaller S, Poc P, Lewis AH, Oh J, Sadighi R, et al. (February 2024). "Covalent bicyclization of protein complexes yields durable quaternary structures". Chem. 10 (2): 615–627. Bibcode:2024Chem...10..615H. doi:10.1016/j.chempr.2023.10.003. PMC 10857811. PMID 38344167.
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