@article{f69dbd5267cd466688b021249f78d5f5,
title = "A facile approach for the in vitro assembly of multimeric membrane transport proteins",
abstract = " 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 Glt Ph , a glutamate transporter homolog that is trimeric in the native state. We use heteromeric Glt Ph 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. ",
author = "Riederer, {Erika A.} and Focke, {Paul J.} and Georgieva, {Elka R.} and Nurunisa Akyuz and Kimberly Matulef and Borbat, {Peter P.} and Freed, {Jack H.} and Blanchard, {Scott C.} and Olga Boudker and Valiyaveetil, {Francis I.}",
note = "Funding Information: This research was supported by the grants from the NIH: R01 GM087546 (FV), R37NS085318 (OB, SB and FV), P41GM103521 (JHF, ACERT) and R01 GM123779 (JHF and ERG). PJF was supported by a postdoctoral fellowship from the American Heart Association (12POST11910068). National Institute of General Medical Sciences R01 GM087546 Francis I Valiyaveetil. Howard Hughes Medical Institute Olga Boudker. National Institute of Neurological Disorders and Stroke R37 NS085318 Scott C Blanchard Olga Boudker Francis I Valiyaveetil. National Institute of General Medical Sciences P41GM103521 Jack H Freed. National Institute of General Medical Sciences R01 GM123779 Elka R Georgieva Jack H Freed. Funding Information: This research was supported by the grants from the NIH: R01 GM087546 (FV), R37NS085318 (OB, SB and FV), P41GM103521 (JHF, ACERT) and R01 GM123779 (JHF and ERG). PJF was supported by a postdoctoral fellowship from the American Heart Association (12POST11910068). Publisher Copyright: {\textcopyright} Riederer et al.",
year = "2018",
month = jun,
day = "11",
doi = "10.7554/eLife.36478",
language = "English (US)",
volume = "7",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
}