Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein

Manzi, Lucio; Barrow, Andrew S.; Hopper, Jonathan T.S.; Kaminska, Renata; Kleanthous, Colin; Robinson, Carol V.; Moses, John E.; Oldham, Neil J.

Abstract

© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Mapping the interaction sites between membrane-spanning proteins is a key challenge in structural biology. In this study a carbene-footprinting approach was developed and applied to identify the interfacial sites of a trimeric, integral membrane protein, OmpF, solubilised in micelles. The diazirine-based footprinting probe is effectively sequestered by, and incorporated into, the micelles, thus leading to efficient labelling of the membrane-spanning regions of the protein upon irradiation at 349 nm. Areas associated with protein–protein interactions between the trimer subunits remained unlabelled, thus revealing their location.

Citation

Manzi, L., Barrow, A. S., Hopper, J. T., Kaminska, R., Kleanthous, C., Robinson, C. V., …Oldham, N. J. (2017). Carbene Footprinting Reveals Binding Interfaces of a Multimeric Membrane-Spanning Protein. Angewandte Chemie International Edition, 56(47), 14873-14877. https://doi.org/10.1002/anie.201708254

• Journal Article Type: Article
• Acceptance Date: Sep 27, 2017
• Online Publication Date: Oct 20, 2017
• Publication Date: Nov 20, 2017
• Deposit Date: Oct 3, 2017
• Publicly Available Date: Oct 21, 2018
• Journal: Angewandte Chemie - International Edition
• Print ISSN: 1433-7851
• Electronic ISSN: 1521-3773
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 56
• Issue: 47
• Pages: 14873-14877
• DOI: https://doi.org/10.1002/anie.201708254
• Keywords: carbene footprinting; integral membrane proteins; mass spectrometry; protein labelling; protein structures
• Public URL: https://nottingham-repository.worktribe.com/output/895876
• Publisher URL: http://onlinelibrary.wiley.com/doi/10.1002/anie.201708254/abstract?campaign=wolacceptedarticle
• Additional Information: This is the peer reviewed version of the following article: Manzi, L., Barrow, A., Hopper, J., Kaminska, R., Kleanthous, C., Robinson, C., Moses, J. and Oldham, N. J. (), Carbene footprinting reveals binding interfaces of a multimeric membrane spanning protein. Angew. Chem. Int. Ed., which has been published in final form at http://dx.doi.org/10.1002/anie.201708254. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.