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.
| Language | English |
|---|---|
| Pages | 11164-11170 |
| Number of pages | 7 |
| Journal | Angewandte Chemie - International Edition |
| Volume | 57 |
| Issue number | 35 |
| Early online date | 30 May 2018 |
| DOIs | |
| Publication status | Published - 27 Aug 2018 |
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Keywords
- INCYPRO
- protein engineering
- protein labeling
- sortase A
- tertiary structure
Cite this
}
In Situ Cyclization of Native Proteins : Structure-Based Design of a Bicyclic Enzyme. / Pelay-Gimeno, Marta; Bange, Tanja; Hennig, Sven; Grossmann, Tom N.
In: Angewandte Chemie - International Edition, Vol. 57, No. 35, 27.08.2018, p. 11164-11170.Research output: Contribution to Journal › Article › Academic › peer-review
TY - JOUR
T1 - In Situ Cyclization of Native Proteins
T2 - Angewandte Chemie - International Edition
AU - Pelay-Gimeno, Marta
AU - Bange, Tanja
AU - Hennig, Sven
AU - Grossmann, Tom N.
N1 - © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2018/8/27
Y1 - 2018/8/27
N2 - 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.
AB - 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.
KW - INCYPRO
KW - protein engineering
KW - protein labeling
KW - sortase A
KW - tertiary structure
UR - http://www.scopus.com/inward/record.url?scp=85051859201&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051859201&partnerID=8YFLogxK
U2 - 10.1002/anie.201804506
DO - 10.1002/anie.201804506
M3 - Article
VL - 57
SP - 11164
EP - 11170
JO - Angewandte Chemie - International Edition
JF - Angewandte Chemie - International Edition
SN - 1521-3773
IS - 35
ER -