International Journal of Molecular Sciences Review Fake It ‘Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics Johannes Thoma 1,2 and Björn M. Burmann 1,2,* 1 Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, 405 30 Göteborg, Sweden; [email protected] 2 Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Göteborg, Sweden * Correspondence: [email protected]; Tel.: +46-317-863-937 Abstract: Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular mem- branes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The lat- ter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics. Keywords: membrane protein; lipid bilayer; membrane mimetic Citation: Thoma, J.; Burmann, B.M. Fake It ‘Till You Make It—The Pur- 1. Introduction suit of Suitable Membrane Mimetics for Membrane Protein Biophysics. Int. The overwhelming majority of scientific articles on membrane proteins introduces this J. Mol. Sci. 2021, 22, 50. https://dx.doi. class of proteins by mentioning their contribution of roughly 30% to organisms’ genomes, org/10.3390/ijms22010050 thus, highlighting their importance and resulting self-explanatory relevance. In the interest of avoiding a repetition of what has been written countless times before, let us instead Received: 9 December 2020 spend a moment of consideration on the endangered species of the polar bear. The polar Accepted: 19 December 2020 bear (Ursus maritimus) is perfectly adapted to life on the annual sea ice of the arctic circle, Published: 23 December 2020 a very specific habitat, which it roams as wide-ranging predator hunting seals [1]. When po- lar bears are relocated from their natural environment to live in captivity in zoological Publisher’s Note: MDPI stays neu- gardens, they have a high tendency to develop abnormal repetitive behavior, such as stereo- tral with regard to jurisdictional claims typical pacing and head nodding [2], and have severely increased infant mortality rates [3]. in published maps and institutional Similar alterations of behavior and infant mortality in captivity have been reported for affiliations. several different species, whereupon the degree of these alterations is directly correlated to the extent of the environmental difference, but rarely as pronounced as in polar bears [3]. Much like this admittedly farfetched example, membrane proteins evolved to reside in Copyright: © 2020 by the authors. Li- the very specific amphipathic lipid bilayer environment of biological membranes and their censee MDPI, Basel, Switzerland. This removal from this environment often results in pronounced structural and functional rami- article is an open access article distributed fications [4–6]. It is, thus, one of the great challenges of membrane protein biophysics to under the terms and conditions of the characterize membrane proteins, while maintaining the specific nature of their lipid bilayer Creative Commons Attribution (CC BY) environment to be able study this class of proteins in a biologically meaningful context. license (https://creativecommons.org/ The continuously progressing efforts to recreate this environment, in order to facilitate licenses/by/4.0/). the biophysical characterization of membrane proteins are the subject of this review. Int. J. Mol. Sci. 2021, 22, 50. https://dx.doi.org/10.3390/ijms22010050 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 22 Int. J. Mol. Sci. 2021, 22, 50 biologically meaningful context. The continuously progressing efforts to recreate this2 of en- 22 vironment, in order to facilitate the biophysical characterization of membrane proteins are the subject of this review. Lipid bilayers form the physical permeability barriers, which segregate cells and cel- Lipid bilayers form the physical permeability barriers, which segregate cells and lularcellular compartments. compartments. Driven Driven by the by hydrophobic the hydrophobic effect, effect, amphiphilic amphiphilic lipid lipid molecules molecules self- assembleself-assemble to form to form lamellar lamellar bilayers, bilayers, with withtheir hydrophobic their hydrophobic moieties moieties facing facing the core the of core the bilayerof the bilayer and their and hydrophilic their hydrophilic head groups head groupsfacing the facing surrounding the surrounding aqueous aqueous environment envi- [7].ronment Cellular [7]. membranes Cellular membranes are formed arefrom formed a large from variety a largeof chemically variety ofvery chemically diverse lipids very [8],diverse ranging lipids from [8], ranginghundreds from of different hundreds lipid of different species lipid in “simple” species inprokaryotic “simple” prokaryotic organisms likeorganisms Escherichia like coliEscherichia to thousands coli to in thousands more complex in more eukaryotic complex organisms eukaryotic [9,10]. organisms The diverse [9,10]. Thephysiochemical diverse physiochemical properties of different properties cellu oflar different membranes cellular are membranes shaped by their are shapedlipid com- by positiontheir lipid [9]. composition Biological membra [9]. Biologicalnes obtain membranes their functionality obtain their only functionality through the only presence through of specializedthe presence integral of specialized membrane integral proteins, membrane whic proteins,h transmit which molecules, transmit energy molecules, and stimuli energy acrossand stimuli these acrossphysical these barriers. physical To fulfil barriers. these To crucial fulfil thesefunctions crucial membrane functions proteins membrane depend pro- onteins the depend properties on the of propertiesthe surrounding of the surroundingmembrane environment membrane environment [11–13]. Factors, [11–13 such]. Factors, as the lipidsuch ascomposition the lipid composition and bilayer andasymmetry, bilayer asymmetry,membrane curvature, membrane tension curvature, as well tension as the as wellfluidity as theof the fluidity bilayer of thedirectly bilayer impact directly the structural impact the and structural functional and integrity functional of integritymembrane of proteinsmembrane [13–15]. proteins [13–15]. Unfortunately, in in their their native native cellular cellular form, form, membrane membrane proteins proteins immanently immanently defy defy the requirementsthe requirements of biophysical of biophysical experiments, experiments, which which demand demand protein protein samples samples of high of purity high purityand high and concentration, high concentration, often in often form in of form a solution. of a solution. In addition In addition to being to beinginsoluble, insoluble, indi- vidualindividual membrane membrane protein protein species species occur occur in rath in ratherer low lowdensities densities in cellular in cellular membranes. membranes. To Tobridge bridge this this gap gap and and make make membrane membrane proteins proteins experimentally experimentally accessible accessible a a multitude of membrane mimeticsmimetics have have been been developed developed over over the the last last decades. decades. As implied As implied by their by name,their name,membrane membrane mimetics mimetics attempt attempt to imitate to imitate the environment the environment of lipid of bilayers.lipid bilayers. In their In mosttheir mostfundamental fundamental forms, forms, as detergent as detergent micelles, micelles this means, this means simply simply to recreate to recreate the hydrophobic the hydro- phobiccore environment core environment of a lipid of bilayera lipid [bilayer16]. However, [16]. However, more complex more complex forms such forms as bicellessuch as bicellesand nanodiscs and nanodiscs try to incorporate try to incorporate a certain numbera certain of number lipid molecules of lipid molecules (Figure1). Moreover,(Figure 1). Moreover,purified membrane purified membrane proteins can pr beoteins reconstituted can be reconstituted into bilayers into of syntheticbilayers of lipids synthetic or lipid li- pidsextracts, or lipid attempting extracts, to attempting closely resemble to closely the resemble original lipidthe original bilayer lipid a membrane bilayer a protein membrane was proteinpurified was from. purified from. Figure 1.1. Solubilized states of membranemembrane proteins.proteins. (A) MembraneMembrane protein inin aa detergentdetergent micellemicelle (cyan).(cyan). ((B)) MembraneMembrane