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 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.
Highlights
Movement of ions and small molecules across cellular membranes takes place through transport proteins such as ion channels and transporters
To develop a protocol for the in vitro reassembly of multimeric membrane proteins, we initially focused on GltPh, a homo-trimeric sodium coupled aspartate transporter (Figure 1A)(Yernool et al, 2004; Boudker et al, 2007)
We found that reassembled GltPh (r-GltPh) in the presence of 200 mM Na+ and 100 mM Asp, sampled three Fluorescence Resonance Energy Transfer (FRET) efficiency states, low, intermediate- and high-FRET centered at ~0.35, ~0.55 and ~0.8, respectively (Figure 2B)
Summary
Movement of ions and small molecules across cellular membranes takes place through transport proteins such as ion channels and transporters. The presence of multiple identical subunits in transport proteins raises the pertinent question of whether there is a functional coupling or ‘crosstalk’ between the subunits In lieu of complete refolding, we envisioned an approach in which we dissociate the native wild type and mutant multimeric proteins into subunits and use a mixture of the dissociated subunits for assembly of the hetero-oligomers. We anticipated that this dissociation/reassociation approach should be generally applicable and provide higher yields compared to complete refolding. We establish the general applicability of the methodology by carrying out the in vitro reassembly of the archaeal transporter GltSm and the bacterial transporters VcINDY and CLC-ec
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