Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain
Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly [superscript 13]C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | en_US |
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John Wiley & Sons, Inc.
2017
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| Online Access: | http://hdl.handle.net/1721.1/107217 https://orcid.org/0000-0002-1567-9672 https://orcid.org/0000-0002-0022-2637 https://orcid.org/0000-0001-5255-5858 |
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| author | Kwon, Byungsu Tietze, Daniel White, Paul Braden Liao, Shu-Yu Hong, Mei |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Kwon, Byungsu Tietze, Daniel White, Paul Braden Liao, Shu-Yu Hong, Mei |
| author_sort | Kwon, Byungsu |
| collection | MIT |
| description | Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly [superscript 13]C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra-membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly [superscript 13]C-labeled full-length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid-phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post-AH cytoplasmic residues exhibit random-coil chemical shifts, low bond order parameters, and a surface-bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus-mimetic membrane, indicating that the structure and dynamics of the post-AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium-sized membrane proteins to provide site-specific structural constraints, which complement the information obtained from uniformly [superscript 13]C, [superscript 15]N-labeled proteins. |
| first_indexed | 2024-09-23T11:43:59Z |
| format | Article |
| id | mit-1721.1/107217 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:43:59Z |
| publishDate | 2017 |
| publisher | John Wiley & Sons, Inc. |
| record_format | dspace |
| spelling | mit-1721.1/1072172022-09-27T21:33:03Z Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain Kwon, Byungsu Tietze, Daniel White, Paul Braden Liao, Shu-Yu Hong, Mei Massachusetts Institute of Technology. Department of Chemistry Hong, Mei Kwon, Byungsu Tietze, Daniel White, Paul Braden Liao, Shu-Yu Hong, Mei Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly [superscript 13]C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra-membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly [superscript 13]C-labeled full-length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid-phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post-AH cytoplasmic residues exhibit random-coil chemical shifts, low bond order parameters, and a surface-bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus-mimetic membrane, indicating that the structure and dynamics of the post-AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium-sized membrane proteins to provide site-specific structural constraints, which complement the information obtained from uniformly [superscript 13]C, [superscript 15]N-labeled proteins. National Institutes of Health (U.S.) (NIH Grant Number: GM088204) National Institutes of Health (U.S.) (NIH Grant Number: P41-EB-002026) 2017-03-07T17:19:26Z 2017-03-07T17:19:26Z 2015-05 2015-04 Article http://purl.org/eprint/type/JournalArticle 09618368 http://hdl.handle.net/1721.1/107217 Kwon, Byungsu, Daniel Tietze, Paul B. White, Shu Y. Liao, and Mei Hong. “Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain.” Protein Science 24, no. 7 (May 27, 2015): 1087–1099. https://orcid.org/0000-0002-1567-9672 https://orcid.org/0000-0002-0022-2637 https://orcid.org/0000-0001-5255-5858 en_US http://dx.doi.org/10.1002/pro.2690 Protein Science Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf John Wiley & Sons, Inc. Prof. Hong via Erja Kajosalo |
| spellingShingle | Kwon, Byungsu Tietze, Daniel White, Paul Braden Liao, Shu-Yu Hong, Mei Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain |
| title | Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain |
| title_full | Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain |
| title_fullStr | Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain |
| title_full_unstemmed | Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain |
| title_short | Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain |
| title_sort | chemical ligation of the influenza m2 protein for solid state nmr characterization of the cytoplasmic domain |
| url | http://hdl.handle.net/1721.1/107217 https://orcid.org/0000-0002-1567-9672 https://orcid.org/0000-0002-0022-2637 https://orcid.org/0000-0001-5255-5858 |
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