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

Marta Pelay-Gimeno, Tanja Bange, Sven Hennig, Tom N. Grossmann*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)11164-11170
Number of pages7
JournalAngewandte Chemie. International Edition
Issue number35
Early online date30 May 2018
Publication statusPublished - 27 Aug 2018


We thank A. Musacchio (MPI Dortmund) for the SrtA expression construct and D. M. Krüger (CGC Dortmund) for a discussion of SrtA variants. We thank F. Müller (MPI Dortmund) for technical support with mass spectrometry. We thank the Deutsche Forschungsgemeinschaft (DFG; Emmy Noether program GR3592/2-1) and the European Research Council (ERC; ERC starting grant 678623). We are grateful for support by AstraZeneca, Bayer CropScience, Bayer HealthCare, Boehringer Ingelheim, Merck KGaA, and the Max Planck Society.

FundersFunder number
Boehringer Ingelheim
Bayer CropScience
Merck KGaA
Horizon 2020 Framework Programme678623
European Research Council
Bayer HealthCare
Deutsche ForschungsgemeinschaftGR3592/2-1


    • protein engineering
    • protein labeling
    • sortase A
    • tertiary structure


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