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[edit]
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[edit]
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[edit]
- Bioconjugation
- Biotechnology
- Protein aggregation
- Protein folding
- Protein quaternary structure
- Protein tertiary structure
References[edit]
- ^ a b c d 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.
- ^ a b 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.
- ^ 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.
- ^ 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.
- ^ a b 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.
- ^ a b c d 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.