Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

Englert, Markus; Nakamura, Akiyoshi; Wang, Yane-Shih; Eiler, Daniel; Söll, Dieter; Guo, Li-Tao

doi:10.1093/nar/gkv1255

Title: Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

Journal Article · Tue Nov 17 00:00:00 EST 2015 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkv1255· OSTI ID:1241074

Englert, Markus ^[1]; Nakamura, Akiyoshi ^[1]; Wang, Yane-Shih ^[1]; Eiler, Daniel ^[1]; Söll, Dieter ^[1]; Guo, Li-Tao ^[1]

Yale Univ., New Haven, CT (United States)

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-L-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: National Institutes of Health (NIH)

Grant/Contract Number:: GM022854; P01 GM022778; GM22854

OSTI ID:: 1241074

Journal Information:: Nucleic Acids Research, Vol. 43, Issue 22; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 17 works

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