Orthogonal translation with 5‐cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding

Author:

von Freiherr Sass Georg Johannes1,Blain‐Hartung Matthew2,Baumann Tobias1,Forest Katrina T.3,Hildebrandt Peter2,Budisa Nediljko14ORCID

Affiliation:

1. Technische Universität Berlin Institut für Chemie/Biokatalyse Berlin Germany

2. Technische Universität Berlin Institut für Chemie / Physikalische Chemie Berlin Germany

3. Department of Bacteriology University of Wisconsin‐Madison Madison Wisconsin USA

4. Department of Chemistry University of Manitoba Winnipeg Manitoba Canada

Abstract

AbstractOrthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile‐containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi‐rational approach to engineer a tyrosyl‐tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5‐cyanotryptophan (5CNW) via orthogonal translation. We combined one round of the well‐established positive selection system with saturation mutagenesis at preselected TyrRS positions, resulting in a novel 5CNW‐specific enzyme that also exhibits high substrate tolerance to other aromatic noncanonical amino acids. We demonstrated the utility of our orthogonal pair by inserting 5CNW into the cyanobacteriochrome Slr1393g3, a bilin‐binding photosensor of the phytochrome superfamily. The nitrile (CN) group of the inserted 5CNW provides non‐invasive labeling in the local structural context while yielding information on local electrostatics and hydrogen bonding by IR spectroscopy. 5CNW is a versatile probe that can be used for both static and dynamic measurements.

Funder

Einstein Stiftung Berlin

Natural Sciences and Engineering Research Council of Canada

Publisher

Wiley

Subject

Molecular Biology,Biochemistry

Cited by 1 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

1. Triple-Bond Vibrations: Emerging Applications in Energy and Biological Sciences;The Journal of Physical Chemistry Letters;2023-12-29

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