Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer
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imperaliMusial-JACS-2016-FINAL[1].pdf
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Author(s)
Musial-Siwek, Monika
Jaffee, Marcie Beth
Imperiali, Barbara
Date Issued
February 2016
Journal
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Citation
Musial-Siwek, Monika, Marcie B. Jaffee, and Barbara Imperiali. “Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer.” Journal of the American Chemical Society 138, no. 11 (March 23, 2016): 3806–3812. doi:10.1021/jacs.5b13426.
Version
Author's final manuscript
Abstract
ntegral membrane proteins play essential roles in all living systems; however, major technical hurdles challenge analyses of this class of proteins. Biophysical approaches that provide structural information to complement and leverage experimentally determined and computationally predicted structures are urgently needed. Herein we present the application of luminescence resonance energy transfer (LRET) for investigating the interactions of the polytopic membrane-bound oligosaccharyl transferases (OTases) with partner substrates. Monomeric OTases, such as the PglBs from Campylobacter jejuni and Campylobacter lari, catalyze transfer of glycans from membrane-associated undecaprenol diphosphate-linked substrates to proteins in the bacterial periplasm. LRET-based distance measurements are enabled by the inclusion of an encoded N-terminal lanthanide-binding tag (LBT), and LRET between the luminescent (LBT)-Tb³⁺ donor complex and fluorescently labeled peptide and glycan substrates provides discrete distance measurements across the span of the membrane. LRET-based measurements of detergent-solubilized PglB from C. lari allowed direct comparison with the distances based on the previously reported the C. lari PglB crystal structure, thereby validating the approach in a defined system. Distance measurements between peptide and glycan substrates and the C. jejuni PglB offer new experimental information on substrate binding to the related, but structurally uncharacterized, eukaryotic OTase.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/jacs.5b13426
