In situ cyclization of proteins
{{Short description|Protein engineering technology}}
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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.{{cite journal | vauthors = Neubacher S, Saya JM, Amore A, Grossmann TN | title = In Situ Cyclization of Proteins (INCYPRO): Cross-Link Derivatization Modulates Protein Stability | journal = The Journal of Organic Chemistry | volume = 85 | issue = 3 | pages = 1476–1483 | date = February 2020 | pmid = 31790232 | pmc = 7011175 | doi = 10.1021/acs.joc.9b02490 }} For such applications, it is essential that the used proteins maintain their structural integrity.{{cite journal | vauthors = Gligorijević V, Renfrew PD, Kosciolek T, Leman JK, Berenberg D, Vatanen T, Chandler C, Taylor BC, Fisk IM, Vlamakis H, Xavier RJ, Knight R, Cho K, Bonneau R | display-authors = 6 | title = Structure-based protein function prediction using graph convolutional networks | journal = Nature Communications | volume = 12 | issue = 1 | pages = 3168 | date = May 2021 | pmid = 34039967 | pmc = 8155034 | doi = 10.1038/s41467-021-23303-9 | bibcode = 2021NatCo..12.3168G }} This is, however, often challenged due to the conditions required for these applications which necessitates protein engineering to stabilize the protein structure.{{cite journal | vauthors = Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K | title = Engineering the third wave of biocatalysis | journal = Nature | volume = 485 | issue = 7397 | pages = 185–194 | date = May 2012 | pmid = 22575958 | doi = 10.1038/nature11117 | bibcode = 2012Natur.485..185B | s2cid = 4379415 }} 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{{cite journal | vauthors = Pelay-Gimeno M, Bange T, Hennig S, Grossmann TN | title = In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme | journal = Angewandte Chemie | volume = 57 | issue = 35 | pages = 11164–11170 | date = August 2018 | pmid = 29847004 | pmc = 6120448 | doi = 10.1002/anie.201804506 }} or protein complex.{{cite journal | vauthors = Hutchins GH, Kiehstaller S, Poc P, Lewis AH, Oh J, Sadighi R, Pearce NM, Ibrahim M, Drienovská I, Rijs AM, Neubacher S, Hennig S, Grossmann TN | display-authors = 6 | title = Covalent bicyclization of protein complexes yields durable quaternary structures | journal = Chem | volume = 10 | issue = 2 | pages = 615–627 | date = February 2024 | pmid = 38344167 | pmc = 10857811 | doi = 10.1016/j.chempr.2023.10.003 | bibcode = 2024Chem...10..615H }} 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.{{cite journal | vauthors = Kiehstaller S, Hutchins GH, Amore A, Gerber A, Ibrahim M, Hennig S, Neubacher S, Grossmann TN | display-authors = 6 | title = Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions | journal = Bioconjugate Chemistry | volume = 34 | issue = 6 | pages = 1114–1121 | date = June 2023 | pmid = 37246906 | pmc = 10288436 | doi = 10.1021/acs.bioconjchem.3c00151 }} 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.