Impact of azidohomoalanine incorporation on protein structure and ligand binding

  • The impact of the incorporation of a non-natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post-translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X-ray crystallography. The structure and dynamics of the apo state of AZH-modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH-modified PDZ3 and an azulene-linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene-linked peptide and the isotopically labelled protein. Co-crystallisation and soaking failed for the peptide-bound holo complex. NMR spectroscopy, however, allowed determination of the protein-ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA-modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target.

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Author:Florian Lehner, Denis Kudlinzki, Christian RichterORCiD, Henrike Müller-Werkmeister, Katharina Barbara Eberl, Jens Bredenbeck, Harald SchwalbeORCiDGND, Robert Silvers
Pubmed Id:
Parent Title (English):Postprint, zuerst in: ChemBioChem
Document Type:Article
Year of Completion:2019
Date of first Publication:2017/09/26
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2019/09/25
Tag:NMR spectroscopy; X-ray diffraction; amino acids; proteins; structure elucidation
Page Number:11
First Page:2340
Last Page:2350
Postprint, zuerst in: ChemBioChem 18.2017, 23, S. 2340-2350, doi: 10.1002/cbic.201700437
Gefördert durch: European Union: Horizon 2020. Infrastructure for NMR, EM and X-rays for Translational Research, iNEXT, H2020 Grant # 653706
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Licence (German):License LogoDeutsches Urheberrecht