A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates

Abstract

Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.

Inducing conformational preference of the membrane protein transporter EmrE through conservative mutations

“…To determine the effect of drug binding on the conformational equilibrium, we acquired NMR spectra for the mixed dimers in the presence of ethidium and tetraphenylphosphonium. These drug substrates are commonly used in the EmrE literature for measuring resistance, transport, and binding (Yerushalmi et al, 1995; Curnow et al, 2004; Robinson et al, 2018). Addition of ethidium induced a significant amount of spectral broadening and ablation of peak intensities in both the wild-type sample and that of the mixed dimer where wild-type was isotopically enriched (Figure 3C).…”

Highly coupled transport can be achieved in free-exchange transport models

“…In buffered drug-monitored liposomal transport assays (Robinson et al, 2018) and in live cells (Bakker and Mangerich, 1981), the proton motive force remains relatively constant. Therefore, we make the simplifying assumption of infinite buffering, such that internal/external proton concentration remains constant throughout the simulation.…”

Cooperative Gating of a K+ Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy

“…Membrane proteins perform vital tasks that must be tightly regulated, including cellular molecular transport and signal transduction . Many membrane proteins are directly activated, inactivated, or otherwise regulated by specific lipid types, driving dynamic conformational exchange in their native lipid environments .…”