A facile approach for the in vitro assembly of multimeric membrane transport proteins
Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2018-06-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/36478 |
| _version_ | 1818027043266232320 |
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| author | Erika A Riederer Paul J Focke Elka R Georgieva Nurunisa Akyuz Kimberly Matulef Peter P Borbat Jack H Freed Scott C Blanchard Olga Boudker Francis I Valiyaveetil |
| author_facet | Erika A Riederer Paul J Focke Elka R Georgieva Nurunisa Akyuz Kimberly Matulef Peter P Borbat Jack H Freed Scott C Blanchard Olga Boudker Francis I Valiyaveetil |
| author_sort | Erika A Riederer |
| collection | DOAJ |
| description | Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter. |
| first_indexed | 2024-12-10T04:41:37Z |
| format | Article |
| id | doaj.art-39e3a06096de4aa7b5b9638d479f75a6 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-12-10T04:41:37Z |
| publishDate | 2018-06-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-39e3a06096de4aa7b5b9638d479f75a62022-12-22T02:01:52ZengeLife Sciences Publications LtdeLife2050-084X2018-06-01710.7554/eLife.36478A facile approach for the in vitro assembly of multimeric membrane transport proteinsErika A Riederer0https://orcid.org/0000-0003-1011-6536Paul J Focke1Elka R Georgieva2Nurunisa Akyuz3Kimberly Matulef4https://orcid.org/0000-0002-5011-9064Peter P Borbat5Jack H Freed6https://orcid.org/0000-0003-4288-2585Scott C Blanchard7https://orcid.org/0000-0003-2717-9365Olga Boudker8https://orcid.org/0000-0001-6965-0851Francis I Valiyaveetil9https://orcid.org/0000-0002-3387-8018Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, United StatesDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, United StatesDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, Unites States; National Biomedical Center for Advanced Electron Spin Resonance Technology, Cornell University, Ithaca, United StatesWeill Cornell Medicine, New York, United StatesDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, United StatesDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, Unites States; National Biomedical Center for Advanced Electron Spin Resonance Technology, Cornell University, Ithaca, United StatesDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, Unites States; National Biomedical Center for Advanced Electron Spin Resonance Technology, Cornell University, Ithaca, United StatesWeill Cornell Medicine, New York, United StatesWeill Cornell Medicine, New York, United States; Howard Hughes Medical Institute, Maryland, United StatesDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, United StatesMembrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.https://elifesciences.org/articles/36478membrane proteinsin vitro foldingDEERFRETmultimerizationtransporters |
| spellingShingle | Erika A Riederer Paul J Focke Elka R Georgieva Nurunisa Akyuz Kimberly Matulef Peter P Borbat Jack H Freed Scott C Blanchard Olga Boudker Francis I Valiyaveetil A facile approach for the in vitro assembly of multimeric membrane transport proteins eLife membrane proteins in vitro folding DEER FRET multimerization transporters |
| title | A facile approach for the in vitro assembly of multimeric membrane transport proteins |
| title_full | A facile approach for the in vitro assembly of multimeric membrane transport proteins |
| title_fullStr | A facile approach for the in vitro assembly of multimeric membrane transport proteins |
| title_full_unstemmed | A facile approach for the in vitro assembly of multimeric membrane transport proteins |
| title_short | A facile approach for the in vitro assembly of multimeric membrane transport proteins |
| title_sort | facile approach for the in vitro assembly of multimeric membrane transport proteins |
| topic | membrane proteins in vitro folding DEER FRET multimerization transporters |
| url | https://elifesciences.org/articles/36478 |
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