Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements: Engineered, Highly Reactive Substrates of Microbial Transglutaminase

Rachel, Natalie M.; Quaglia, Daniela; L�vesque, �ric; Charette, Andr� B.; Pelletier, Joelle N.

Abstract

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side‐chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site‐specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100‐fold increase in incorporation of a recently developed aminated benzo[a ]imidazo[2,1,5‐cd ]indolizine‐type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α‐helical, β‐sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q‐GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG‐reactive glutamines can be readily introduced into a protein domain for fluorescent labeling.

Citation

Rachel, N. M., Quaglia, D., Lévesque, É., Charette, A. B., & Pelletier, J. N. (2017). Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements: Engineered, Highly Reactive Substrates of Microbial Transglutaminase. Protein Science, 26(11), 2268-2279. https://doi.org/10.1002/pro.3286

• Journal Article Type: Article
• Acceptance Date: Aug 28, 2017
• Online Publication Date: Oct 25, 2017
• Publication Date: 2017-11
• Deposit Date: Jun 18, 2020
• Publicly Available Date: Jan 21, 2021
• Journal: Protein Science
• Print ISSN: 0961-8368
• Electronic ISSN: 1469-896X
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 26
• Issue: 11
• Pages: 2268-2279
• DOI: https://doi.org/10.1002/pro.3286
• Public URL: https://nottingham-repository.worktribe.com/output/4672387
• Publisher URL: https://onlinelibrary.wiley.com/doi/full/10.1002/pro.3286
• Additional Information: This is the peer reviewed version of the following article: Rachel, N.M., Quaglia, D., Lévesque, É., Charette, A.B. and Pelletier, J.N. (2017), Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements. Protein Science, 26: 2268-2279, which has been published in final form at https://doi.org/10.1002/pro.3286. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions