Detergent screening of the human voltage‐gated proton channel using fluorescence‐detection size‐exclusion chromatography

Abstract

The human voltage-gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino-and carboxy-termini. The protein is activated by membrane depolarization, similar to other voltage-sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three-dimensional structures for homologues of this voltage-gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino-terminus of the human Hv1 proton channel (termed EGFP-Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence-detection size-exclusion chromatography. The EGFP-Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3-12 and the nonionic n-dodecyl-b-D-maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, Published by Wiley-Blackwell. V C 2014 The Protein Society which is distinguishable from protein aggregates, at the final size-exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full-length human Hv1 proton channel.

Discovery of Functional Antibodies Targeting Ion Channels

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The best of both worlds: A new lipid complex has micelle and bicelle-like properties

“…Bicelles are of unique interest for membrane protein studies because unlike their detergent counterpart, they contain a bilayered planar region, which captures a true membrane-like environment (Figure 1) [4, 5]. In membrane protein studies, choosing the optimal detergent or lipid system usually necessitates a screening of potential candidates because not all systems are universally adaptable to all types of membrane proteins [10 – 13]. In this study, a novel lipid-detergent complex was prepared with DMPC, the long-chain phospholipid, and a nonionic detergent, pentaethylene glycol n-octyl ether or C 8 E 5 (Figure 1).…”