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

Angewandte Chemie
Marta Pelay-GimenoTom N Grossmann

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.

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Citations

Nov 10, 2018·Angewandte Chemie·Carolin Mueller, Tom N Grossmann
Jul 30, 2020·Protein Engineering, Design & Selection : PEDS·Magdalena WójcikYkelien L Boersma
Jul 26, 2019·Chemical Science·Marc-André KasperChristian P R Hackenberger
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Apr 29, 2020·Angewandte Chemie
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Feb 12, 2019·Chembiochem : a European Journal of Chemical Biology·Partha Sarathi AddyAbhishek Chatterjee
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Mar 16, 2021·The Journal of Organic Chemistry·Tyler F HigginsJeffrey D Winkler
Jun 2, 2021·Chembiochem : a European Journal of Chemical Biology·Anissa HaimTom N Grossmann
Oct 6, 2020·Journal of Medicinal Chemistry·Dan SindhikaraAndreas Evers
Dec 4, 2019·The Journal of Organic Chemistry·Saskia NeubacherTom N Grossmann

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Methods Mentioned

BETA
glycosylation
chemical stress
NMR
Fluorescence
fluorescence imager

Software Mentioned

INCYPRO

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