Biology:In situ cyclization of proteins
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.[1][2] For such applications, it is essential that the used proteins maintain their structural integrity.[3] This is, however, often challenged due to the conditions required for these applications which necessitates protein engineering to stabilize the protein structure.[4] 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.[5]
Technology
The INCYPRO technology utilizes tris-reactive molecules to crosslink three defined positions within a protein[1] or protein complex.[6] 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.[1][6] So far, the melting temperature of proteins was increased by up to 39°C in a single design step.[6]
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.[1][5] 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.[2] In addition, a number of homo-trimeric protein complexes was stabilized including the Pseudomonas fluorescens esterase (PFE) and an Enoyl-CoA hydratase.[6] In these cases, enzyme conjugates with overall bicyclic topology were generated.
See also
- Bioconjugation
- Biotechnology
- Protein aggregation
- Protein folding
- Protein quaternary structure
- Protein tertiary structure
References
- ↑ 1.0 1.1 1.2 1.3 "In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme". Angewandte Chemie 57 (35): 11164–11170. August 2018. doi:10.1002/anie.201804506. PMID 29847004.
- ↑ 2.0 2.1 "In Situ Cyclization of Proteins (INCYPRO): Cross-Link Derivatization Modulates Protein Stability". The Journal of Organic Chemistry 85 (3): 1476–1483. February 2020. doi:10.1021/acs.joc.9b02490. PMID 31790232.
- ↑ "Structure-based protein function prediction using graph convolutional networks". Nature Communications 12 (1): 3168. May 2021. doi:10.1038/s41467-021-23303-9. PMID 34039967. Bibcode: 2021NatCo..12.3168G.
- ↑ "Engineering the third wave of biocatalysis". Nature 485 (7397): 185–194. May 2012. doi:10.1038/nature11117. PMID 22575958. Bibcode: 2012Natur.485..185B.
- ↑ 5.0 5.1 "Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions". Bioconjugate Chemistry 34 (6): 1114–1121. June 2023. doi:10.1021/acs.bioconjchem.3c00151. PMID 37246906.
- ↑ 6.0 6.1 6.2 6.3 "Covalent bicyclization of protein complexes yields durable quaternary structures". Chem 10 (2): 615–627. February 2024. doi:10.1016/j.chempr.2023.10.003. PMID 38344167. Bibcode: 2024Chem...10..615H.
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