**1. Introduction**

The overwhelming majority of scientific articles on membrane proteins introduces this class of proteins by mentioning their contribution of roughly 30% to organisms' genomes, 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 spend a moment of consideration on the endangered species of the polar bear. The polar bear (*Ursus maritimus*) is perfectly adapted to life on the annual sea ice of the arctic circle, a very specific habitat, which it roams as wide-ranging predator hunting seals [1]. When polar bears are relocated from their natural environment to live in captivity in zoological gardens, they have a high tendency to develop abnormal repetitive behavior, such as stereotypical pacing and head nodding [2], and have severely increased infant mortality rates [3]. Similar alterations of behavior and infant mortality in captivity have been reported for 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 the very specific amphipathic lipid bilayer environment of biological membranes and their removal from this environment often results in pronounced structural and functional ramifications [4–6]. It is, thus, one of the grea<sup>t</sup> challenges of membrane protein biophysics to characterize membrane proteins, while maintaining the specific nature of their lipid bilayer environment to be able study this class of proteins in a biologically meaningful context. The continuously progressing efforts to recreate this environment, in order to facilitate the biophysical characterization of membrane proteins are the subject of this review.

**Citation:** Thoma, J.; Burmann, B.M. Fake It 'Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics. *Int. J. Mol. Sci.* **2021**, *22*, 50. https://dx.doi. org/10.3390/ijms22010050

Received: 9 December 2020 Accepted: 19 December 2020 Published: 23 December 2020

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Lipid bilayers form the physical permeability barriers, which segregate cells and cellular compartments. Driven by the hydrophobic effect, amphiphilic lipid molecules self-assemble to form lamellar bilayers, with their hydrophobic moieties facing the core of the bilayer and their hydrophilic head groups facing the surrounding aqueous environment [7]. Cellular membranes are formed from a large variety of chemically very diverse lipids [8], ranging from hundreds of different lipid species in "simple" prokaryotic organisms like *Escherichia coli* to thousands in more complex eukaryotic organisms [9,10]. The diverse physiochemical properties of different cellular membranes are shaped by their lipid composition [9]. Biological membranes obtain their functionality only through the presence of specialized integral membrane proteins, which transmit molecules, energy and stimuli across these physical barriers. To fulfil these crucial functions membrane proteins depend on the properties of the surrounding membrane environment [11–13]. Factors, such as the lipid composition and bilayer asymmetry, membrane curvature, tension as well as the fluidity of the bilayer directly impact the structural and functional integrity of membrane proteins [13–15].

Unfortunately, in their native cellular form, membrane proteins immanently defy the requirements of biophysical experiments, which demand protein samples of high purity and high concentration, often in form of a solution. In addition to being insoluble, individual membrane protein species occur in rather low densities in cellular membranes. To bridge this gap and make membrane proteins experimentally accessible a multitude of membrane mimetics have been developed over the last decades. As implied by their name, membrane mimetics attempt to imitate the environment of lipid bilayers. In their most fundamental forms, as detergent micelles, this means simply to recreate the hydrophobic core environment of a lipid bilayer [16]. However, more complex forms such as bicelles and nanodiscs try to incorporate a certain number of lipid molecules (Figure 1). Moreover, purified membrane proteins can be reconstituted into bilayers of synthetic lipids or lipid extracts, attempting to closely resemble the original lipid bilayer a membrane protein was purified from.

**Figure 1.** Solubilized states of membrane proteins. ( **A**) Membrane protein in a detergent micelle (cyan). (**B**) Membrane protein in a disc-shaped bicelle containing lipids (blue). ( **C**) Membrane proteins in nanodiscs stabilized by membrane scaffold protein (MSP, green) and amphipathic polymers (purple).

Naturally, all these methods have their pros and cons and not all of them are compatible with different biophysical methods. For example, solution NMR (Nuclear magnetic resonance) spectroscopy, single-particle cryo-EM (electron microscopy), and X-ra/neutron solution scattering methods demand membrane proteins in a solubilized form, typically in the form of micelles, bicelles or nanodiscs. Solid-state-NMR, negative stain EM, and AFM (atomic force microscopy) are suitable for larger membrane assemblies such as proteoliposomes, whereas X-Ray crystallography requires samples in a three-dimensional (3D) crystalline form. The hydrophobic contacts required to maintain the structural integrity of membrane proteins within these crystals can either be satisfied by detergents or in case of lipidic cubic phases (LCP) by lipid molecules. In this review, we survey the currently available membrane mimetic systems, weigh their advantages, as well as their disadvantages, evaluate how they impact the structural and functional states of membrane proteins and assess their suitability for various biophysical methods.
