Different Purification Approaches for the Integral Membrane Protein EmrE Leads to Biochemical and Biophysical Differences in the Protein

Abstract

EmrE is an integral membrane protein in Escherichia coli belonging to a class of multidrug resistance transporters: the Small Multidrug Resistance transporter family (SMR). EmrE effluxes quaternary cationic compounds (QCC) using a proton motive force. Many groups studying this protein utilize a C-terminal myc-eptitope- 6 histidine tag engineered into EmrE for the purpose of protein purification in a detergent-containing aqueous environment. An alternative method for EmrE purification involves using a solvent mixture of chloroform:methanol:water to extract the protein from bacterial membranes. The EmrE isolated using the latter method does not have any tag. As there is different data appearing from different labs, the goal of this research is to compare the protein purified in with the different approaches analyzed under identical conditions. Untagged and tagged EmrE was analyzed and compared by fluorescence spectroscopy. Fluorescence emission spectra of untagged EmrE revealed a lower combined fluorescence intensity of the tyrosine and tryptophan fluorescence compared to tagged EmrE. A red edge excitation shift (REES) effect was also observed for tagged EmrE indicating that there is a population of fluorophores that have a slower rate of solvent relaxation, unlike the fluorophores in untagged EmrE. Functional analysis of the EmrE by QCC ligand-binding (Ethidium, methyl viologen, cetylpyridinium) was investigated using fluorescence quenching binding curves. Untagged EmrE and tagged EmrE binding curves with ethidium were similar, whereas binding of the methyl viologen and cetylpyridinium were different between untagged and tagged EmrE. These results show that the two purification approaches lead to differences in the structural and function states of this integral membrane protein.