Issue 4, 2017

Mapping a protein recognition centre with chiral photoactive ligands. An integrated approach combining photophysics, reactivity, proteomics and molecular dynamics simulation studies

Abstract

A multidisciplinary strategy to obtain structural information on the intraprotein region is described here. As probe ligands, (S)- and (R)-CPFMe (the methyl esters of the chiral drug carprofen) have been selected, while bovine α1-acid glycoprotein (BAAG) has been chosen as a biological host. The procedure involves the separate irradiation of the BAAG/(S)-CPFMe and BAAG/(R)-CPFMe complexes, coupled with fluorescence spectroscopy, laser flash photolysis, proteomic analysis, docking and molecular dynamics simulations. Thus, irradiation of the BAAG/CPFMe complexes at λ = 320 nm was followed by fluorescence spectroscopy. The intensity of the emission band obtained after irradiation indicated photodehalogenation, whereas its structureless shape suggested covalent binding of the resulting radical CBZMe˙ to the biopolymer. After gel filtration chromatography, the spectra still displayed emission, in agreement with covalent attachment of CBZMe˙ to BAAG. Stereodifferentiation was observed in this process. After trypsin digestion and ESI-MS/MS, the incorporation of CBZMe was detected at Phe68. Docking and molecular dynamics simulation studies, which were carried out using a homology model of BAAG, reveal that the closer proximity of the aromatic moiety of the (S)-enantiomer to the phenyl group of Phe68 would be responsible for the experimentally observed, more effective chemical modification of the protein. The proposed tridimensional structure of BAAG covalently modified by the two enantiomers is also provided. In principle, this approach can be extended to a variety of protein/ligand complexes.

Graphical abstract: Mapping a protein recognition centre with chiral photoactive ligands. An integrated approach combining photophysics, reactivity, proteomics and molecular dynamics simulation studies

Supplementary files

Article information

Article type
Edge Article
Submitted
04 Nov 2016
Accepted
30 Dec 2016
First published
05 Jan 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2017,8, 2621-2628

Mapping a protein recognition centre with chiral photoactive ligands. An integrated approach combining photophysics, reactivity, proteomics and molecular dynamics simulation studies

D. Limones-Herrero, R. Pérez-Ruiz, E. Lence, C. González-Bello, M. A. Miranda and M. C. Jiménez, Chem. Sci., 2017, 8, 2621 DOI: 10.1039/C6SC04900A

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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