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Angew Chem Int Ed Engl. 2018 Aug 27;57(35):11164-11170. doi: 10.1002/anie.201804506. Epub 2018 Jun 21.

In Situ Cyclization of Native Proteins: Structure-Based Design of a Bicyclic Enzyme.

Author information

1
Department of Chemistry & Pharmaceutical Sciences, VU University Amsterdam, De Boelelaan 1108, 1081 HZ, Amsterdam, The Netherlands.
2
Department of Mechanistic Cell Biology, Max-Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227, Dortmund, Germany.
3
Department for Systems Chronobiology, LMU Munich, Goethe-Str. 31, 80336, Munich, Germany.

Abstract

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface-exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24 °C increased thermal stability.

KEYWORDS:

INCYPRO; protein engineering; protein labeling; sortase A; tertiary structure

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