Close allies in membrane protein research: Cell-free synthesis and nanotechnology

Abstract

Membrane proteins control fundamental processes that are inherent to nearly all forms of life such as transport of molecules, catalysis, signaling, vesicle fusion, sensing of chemical and physical stimuli from the environment, and cell-cell interactions. Membrane proteins are harbored within a non-equilibrium fluid-like environment of biological membranes that separate cellular and non-cellular environments, as well as in compartmentalized cellular organelles. One of the classes of membrane proteins that will be specifically treated in this article are transport proteins of plant origin, that facilitate material and energy transfer at the membrane boundaries. These proteins import essential nutrients, export cellular metabolites, maintain ionic and osmotic equilibriums and mediate signal transduction. The aim of this article is to report on the progress of membrane protein functional and structural relationships, with a focus on producing stable and functional proteins suitable for structural and biophysical studies. We interlink membrane protein production primarily through wheat-germ cell-free protein synthesis (WG-CFPS) with the growing repertoire of membrane mimicking environments in the form of lipids, surfactants, amphipathic surfactant polymers, liposomes and nanodiscs that keep membrane proteins soluble. It is hoped that the advancements in these fields could increase the number of elucidated structures, in particular those of plant membrane proteins, and contribute to bridging of the gap between structures of soluble and membrane proteins, the latter being comparatively low.

Amphipols for Each Season

“…It can be either left to precipitate, and then solubilized with a detergent, complexed by a detergent during synthesis, or integrated into lipid vesicles or NDs (see e.g., Etzkorn et al 2013; Katzen et al 2009; Klammt et al 2006, 2011; Lyukmanova et al 2012; Park et al 2007; Shadiac et al 2013; Shenkarev et al 2013, and references therein). Because of their mild character and ability to facilitate folding, APols are an attractive medium into which to let newly expressed MPs fold.…”

Plant High-Affinity Potassium (HKT) Transporters Involved in Salinity Tolerance: Structural Insights to Probe Differences in Ion Selectivity

“…Protein structure determines protein function [18,72,73]. Therefore, the 3D structure of a protein provides vital information as to how a protein functions [73].…”

“…Despite this importance, structural knowledge of IMPs lags behind that of soluble proteins, with less than 400 unique structures of IMP resolved as of December 2012 [16,17]. Notably, fewer than ten of these 400 unique structures are from plants [18]. …”

“…The scarcity of structural knowledge about IMPs is due to the additional challenges associated with obtaining IMP structures as opposed to determining the structures of soluble proteins [2,16,18]. These include: (i) the difficulty in obtaining sufficient amounts of IMPs from native or recombinant sources; (ii) recovering IMPs from membranes without denaturing them; (iii) frequent cytotoxicity issues when over-expressing IMPs in recombinant systems and (iv) the complex crystallisation conditions of IMPs.…”

One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces

“…Consequently, detergents and membrane mimetics (3) are used to solubilize membrane proteins. To avoid cytotoxicity caused by in vivo expression and enable highthroughput production, cell-free systems are used to express membrane proteins in vitro (4)(5)(6)(7). Despite recent progresses, understanding the functions of membrane proteins is still a lengthy and difficult process.…”