Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer
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 structure...
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| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2017
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| Online Access: | http://hdl.handle.net/1721.1/108017 https://orcid.org/0000-0002-7465-2277 https://orcid.org/0000-0002-5749-7869 |
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| author | Musial-Siwek, Monika Jaffee, Marcie Beth Imperiali, Barbara |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Musial-Siwek, Monika Jaffee, Marcie Beth Imperiali, Barbara |
| author_sort | Musial-Siwek, Monika |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T15:58:31Z |
| format | Article |
| id | mit-1721.1/108017 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:58:31Z |
| publishDate | 2017 |
| publisher | American Chemical Society (ACS) |
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| spelling | mit-1721.1/1080172022-09-29T17:25:46Z Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer Musial-Siwek, Monika Jaffee, Marcie Beth Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Imperiali, Barbara Musial-Siwek, Monika Jaffee, Marcie Beth Imperiali, Barbara 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. United States. National Institutes of Health (GM-039334) United States. National Institutes of Health (T32GM007287) National Science Foundation (U.S.) (MCB 0744415) 2017-04-10T17:44:16Z 2017-04-10T17:44:16Z 2016-02 2015-12 Article http://purl.org/eprint/type/JournalArticle 0002-7863 1520-5126 http://hdl.handle.net/1721.1/108017 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. https://orcid.org/0000-0002-7465-2277 https://orcid.org/0000-0002-5749-7869 en_US http://dx.doi.org/10.1021/jacs.5b13426 Journal of the American Chemical Society Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) Prof. Imperiali via Courtney Crummett |
| spellingShingle | Musial-Siwek, Monika Jaffee, Marcie Beth Imperiali, Barbara Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer |
| title | Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer |
| title_full | Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer |
| title_fullStr | Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer |
| title_full_unstemmed | Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer |
| title_short | Probing Polytopic Membrane Protein–Substrate Interactions by Luminescence Resonance Energy Transfer |
| title_sort | probing polytopic membrane protein substrate interactions by luminescence resonance energy transfer |
| url | http://hdl.handle.net/1721.1/108017 https://orcid.org/0000-0002-7465-2277 https://orcid.org/0000-0002-5749-7869 |
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