Abstract
Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this 'one size fits all' method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during 'on-column', 'in-gel', and 'on-bead' reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.
Highlights
Membrane proteins play essential roles, such as membrane transport, signal transduction, cell homeostasis, and energy metabolism
The NSPr peptide was mixed with MalFGK2 in dodecyl maltoside and the mixture applied immediately onto a size exclusion column equilibrated in a detergent-free buffer (Figure 2A)
The 2D-class averages revealed a structure very similar to MalFGK2 in nanodiscs, (Fabre et al, 2017) with distinctly visible elements such as the MalK2 dimer, the periplasmic P2 loop and a larger discoidal density corresponding to the NSPr peptides wrapping around the MalFG membrane domain
Summary
Membrane proteins play essential roles, such as membrane transport, signal transduction, cell homeostasis, and energy metabolism. Membrane proteins are generally purified in detergent micelles, but these small amphipathic molecules are quite often detrimental to protein structure and activity, in addition to interfering with downstream analytical methods This drawback has led researchers to develop detergent-free alternatives such as amphipols (Popot, 2010), SMALPs (Lee et al, 2016), saposin-lipoparticles (Frauenfeld et al, 2016), and the popular nanodisc system (Bayburt et al, 2006; Denisov et al, 2004).
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