Impact of Azidohomoalanine Incorporation on Protein Structure and Ligand Binding. (7th November 2017)

• Record Type: Journal Article
• Authors: Lehner, Florian; Kudlinzki, Denis; Richter, Christian; Müller‐Werkmeister, Henrike M.; Eberl, Katharina B.; Bredenbeck, Jens; Schwalbe, Harald; Silvers, Robert
• Is Part Of: Chembiochem. Volume 18:Number 23(2017)
• Page Start: 2340
• Page End: 2350
• Publication Date: 2017-11-07
• Subjects: amino acids -- NMR spectroscopy -- proteins -- structure elucidation -- X-ray diffraction; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201700437
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: 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. Abstract : Unnatural influence : Unnatural amino acids are widely used to study proteins. NMR spectroscopy and X‐ray diffraction are applied to characterize the structural and dynamic impact of azidohomoalanine (AZH) incorporation into the model protein PDZ3 domain. The structure and dynamics of the apo state of AZH‐modified PDZ3 remains mostly unperturbed.