ACERT Highlights: ACERT's Service and Collaborative Projects

National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

ACERT's Service and Collaborative Projects

A Facile Approach for the in vitro Assembly of Multimeric Membrane Transport Proteins for DEER Spectroscopy

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. We developed a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We established 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 demonstrated 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.

Funding: R01GM087546 (FIV); Howard Hughes Medical Institute (OB); R37NS085318 (SCB, OB, FIV); P41GM103521 (JHF); Amer. Heart Assoc. 12POST1910068 (PJF); R01123779 (JHF, ERG).

Publication: eLife 7, e36478 (2018); PMCID: PMC6025958

Using a heterotrimeric GltPh to probe movements of the transport domain using DEER. (A) A homomeric and a heteromeric GltPh transporter are shown in ribbon representation. The scaffold domain is colored in wheat while the transport domain is colored in light blue. The Cb atoms of the Cys residues at 216 and 294 labelled with spin probes are shown as red spheres. All subunits in the homomeric GltPh carry the Cys substitutions while only one subunit in the heteromeric GltPh carries the Cys substitutions. The distances monitored in the DEER experiment are indicated by solid lines. (B) Background-corrected DEER amplitude, V(t) vs. evolution time t (left). The data are shown for: GltPh homotrimers with spin labels at both positions in each of the protomers prepared with 200 mM NaCl (black); GltPh heterotrimer with spin-labels just in one of the protomers prepared with either 200 mM NaCl (blue) or 200 mM NaCl/300 mM aspartate (red). Inter-spin distance distributions, P(r), reconstructed from the above DEER data, are plotted in respective colors (right). GltPh heterotrimers show much simpler P(r) as compared to convoluted result for homotrimers.

Erika A Riederer, Paul J Focke (Department of Physiology and Pharmacology, Oregon Health & Science University, Portland)
Elka R Georgieva (ACERT)
Nurunisa Akyuz (Weill-Cornell Medicine, New York City)
Kimberly Matulef (Department of Physiology and Pharmacology, Oregon Health & Science University, Portland)
Peter P Borbat, Jack H Freed (ACERT)
Scott C Blanchard (Weill-Cornell Medicine, New York City)
Olga Boudker (Weill-Cornell Medicine, New York City, Howard Hughes Medical Institute, Maryland)
Francis I Valiyaveetil (Department of Physiology and Pharmacology, Oregon Health & Science University, Portland)

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

National Biomedical Resource for Advanced ESR Spectroscopy

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