J.L. Robertson’s present address is the Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, MO.
Copyright © 2018 RobertsonThis article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
Robertson recounts the historical experiments that gave rise to our current understanding of cell membranes.
In 1918, the year the Journal of General Physiology was founded, there was little understanding of the structure of the cell membrane. It was evident that cells had invisible barriers separating the cytoplasm from the external solution. However, it would take decades before lipid bilayers were identified as the essential constituent of membranes. It would take even longer before it was accepted that there existed hydrophobic proteins that were embedded within the membrane and that these proteins were responsible for selective permeability in cells. With a combination of intuitive experiments and quantitative thinking, the last century of cell membrane research has led us to a molecular understanding of the structure of the membrane, as well as many of the proteins embedded within. Now, research is turning toward a physical understanding of the reactions of membrane proteins and lipids in this unique and incredibly complex solvent environment.
IntroductionOrganisms live and thrive in a wide range of chemical environments with varying physical conditions. Life has been found at freezing temperatures, thermal vents, extreme acidic and alkaline conditions, and a wide range of salt concentrations. This diversity is possible because the cellular membrane presents a robust barrier to the external world, setting the stage for exquisite control of the cell’s internal solutions. First and foremost, the membrane must be a barrier to salts, protons, and charged molecules to maintain proper biochemistry. In addition, if the membrane is capable of separating charge, like a low dielectric insulator, then it can also store electrical as well as chemical potential energy to fuel future biological work. Finally, the membrane must be capable of changing its shape to accommodate the increasingly complex requirements of biology, and therefore, it must be fluid in nature. It is remarkable that all of the physical properties listed above are satisfied by a two-molecule-thick layer of self-assembled oil and even more amazing how proteins have evolved to function in this dramatically different physical environment. The lipid bilayer provides a robust solution for a physiological barrier, and its simplicity is a foundation for the origins of life and evolutionary diversity (Schrum et al., 2010).
In this Milestones article, I review some of the key experiments that led to our current understanding of the cell membrane as a lipid bilayer that solvates proteins that span the membrane. It is a daunting task to go over this enormous body of work, and so this article presents a mere sampling of the research. For those desiring a deeper review of the history, I refer you to several other resources (Tanford, 2004; Lombard, 2014; Stillwell, 2016). In this paper, I first discuss how our understanding of cell membranes went from invisible boundaries to fluid lipid bilayers. Next, I highlight the path to identifying a new class of membrane proteins that reside and function within hydrophobic lipid bilayers. Finally, I discuss current research of membrane proteins in lipid bilayers, highlighting the key challenges and complexities of studying proteins in a solvent environment that is self-assembling and has its own structural properties and increased chemical complexity. The past 100 years take us from a time when we did not know the lipid bilayer and integral membrane proteins existed to the present, where the structure of membrane proteins, even in the context of the cell membrane, is becoming more and more accessible. Throughout this time, the Journal of General Physiology has been a pivotal resource for the presentation and discussion of research that is the foundation of the field of cell membrane biophysics.
Equipped with a light microscope, Robert Hooke examined the intricate assemblies of numerous biological samples, documenting these close-up observations in his book, Micrographia (Hooke, 1665). One of the samples he examined was a material that was extracted from the underlayer of bark from a cork tree. He observed that the tissue was composed of a network of pores with thin “Interstitia, or walls” reminiscent of the “thin films of Wax in a Honey-comb.” He referred to these pores as “cells” and proposed that other tissues, in both plants and animals, may be organized in a similar fashion. He extensively studied animal structures such as the valves of the heart and veins but did not see any similar cellular compartmentalization. We now know that the reason he could visualize the cellular structures of cork but not animal cells was because he was seeing the dried up cell walls, easily visible by eye using a microscope. But even still, in plant cells, it was apparent that there was another invisible bounding mechanism that encapsulated the cytosolic solution of protein, salts, and small molecules. This was observed in some cells, where the protoplasm appeared to pull away from the cell wall and also contain the vacuoles and organelles within ( Fig. 1 ). What explained this encapsulation? The prevailing theory for hundreds of years was that the protoplasm was dense and colloidal, rich in protein and other biological molecules, which made it behave as a gel (Lombard, 2014). It was proposed that when this gel came into contact with the extracellular solution, there was a hardening at the contact layer, perhaps through aggregation of the colloidal matrix. For a modern-day example of this, consider the popular molecular gastronomy technique known as spherification (Fu et al., 2014). This is a method that is being used in the food industry to produce artificial caviar or the juicy popping bubbles found in bubble tea. These spheres are constructed by dropping solutions containing a sodium salt of the carbohydrate polymer alginate into calcium chloride. At the interface, calcium binds to the alginate and stabilizes the polymer network to form a hardened shell enclosing the liquid of interest ( Fig. 2 A ). Therefore, there is a chemical precedent for this, but the question remained whether this was the actual mechanism of encapsulation in biological cells.
Plasmolysis reveals invisible barriers within living cells. (A and B) A normal Spirogyra cell (A) and the cell during plasmolysis (B). From Overton (1895), Fig. 1 is adapted from Vierteljahresschr. Naturforsch. Ges. Zürich.
A brief history of cellular barriers. (A) The protoplasmic colloid model. The barrier is a hardened shell that forms when the dense colloidal protoplasm makes contact with the extracellular solution. Pictured here is artificial caviar made by the analogous process of spherification. Photo courtesy of J.L. Robertson. (B) The paucimolecular model of Davson and Danielli, where the cell barrier is modeled as a lipid bilayer with a lipoid core flanked by layers of polar and charged proteins. From Danielli and Davson (1935), Fig. 2 B is adapted with permission from the Journal of Cellular Physiology. (C) The unit membrane model of Robertson, indicating the train track–appearing lipid bilayer that forms a continuous membrane around the cell. From Robertson (1981), Fig. 2 C is adapted with permission from the Journal of Cell Biology. (D) The fluid mosaic model of Singer and Nicolson, showing a lipid bilayer with integral membrane proteins responsible for cellular permeability. From Singer and Nicolson (1972), Fig. 2 D is adapted with permission from Science.
In the second half of the 19th century, a simple experimental idea would introduce a new hypothesis about the bounding mechanism of cells (Tanford, 2004; Lombard, 2014; Stillwell, 2016). In 1855, Carl von Nägeli and Karl Cramer reported their observations that plant cells, when placed in hypertonic solution, set by increasing salt or sugar concentrations, would result in the protoplasm shrinking away from the cell wall (von Nägeli and Cramer, 1855). Conversely, when the cell was placed in a hypotonic solution, the cytosol would expand to the point of bursting. They referred to these experiments as plasmolysis studies ( Fig. 1 ) and interpreted the results as a demonstration of a pliable barrier, capable of adapting its shape to the surrounding vessel: in this case, the cell wall. Finally, they demonstrated that vacuoles, when isolated from the cell, followed the same behavior, and the volume changes here were reversible. Wilhelm Pfeffer continued these studies and proposed that a skin, or plasma membrane, existed that covered the exposed surface of the protoplasm (Pfeffer, 1877). At that time, he suggested this membrane was similar to artificial copper ferrocyanide membranes that had been recently discovered by Moritz Traube (Traube, 1867). These were simple studies, ones that any young student can carry out today, yet the implications of the findings were profound. The results provided evidence that the barrier acted like an invisible skin, selectively allowing for the passage of water in and out of the cell, although limiting the permeability of salts and sugars.
The early days of plasmolysis research were limited to qualitative visualization of changes under a microscope. Some studies aimed to quantify the changes by isolating the cells and weighing them before and after the osmotic stress, which sounds like a challenging task. However, Charles E. Overton would contribute significantly to our understanding of the membrane barrier by applying the osmometric technique with the understanding that the total osmotic pressure is equal to the sum of partial pressures in solution (Kleinzeller, 1999). With this improved resolution in hand, he could apply a quantitative and extensive approach to plasmolysis studies. Over a short period of time, from 1890 to 1900, he studied nearly 500 different compounds for setting the osmotic gradient, including salts, sugars, and nonpolar molecules (Overton, 1895; Kleinzeller, 1997, 1999). One of the surprising findings was that aliphatic alcohols did not lead to changes in the protoplasm’s volume. Using his large body of quantitative data, he built a physical model of the cell membrane to explain his observations, modeled after gas laws that were being developed at the same time. He proposed that the lack of the change in the cell volume was caused by the free equilibration of nonpolar or lipoid-like molecules across the cell membrane. With this, he postulated that the cell membrane itself must be composed of similar nonpolar molecules to support the free partitioning of these substances and at the same time provide a barrier to salts and sugars. Therefore, the cell membrane was lipid impregnated and a distinctly different chemical environment from that of the cell’s protoplasm or the external solution in which the cell bathed. Most surprisingly, he even proposed that cholesterol and phospholipids could be candidates for the nonpolar chemicals composing the membrane. Finally, he investigated a series of nonpolar molecules that were well known for their activity in humans: general anesthetics. In parallel research with Hans Horst Meyer, they both found that the propensity for an anesthetic to partition into olive oil strongly correlated with its efficacy in the clinical setting (Meyer, 1899; Overton, 1901), supporting the idea of a lipid-filled nonpolar barrier to cells ( Fig. 3 ). The work of Overton and Meyer is a fine example of biophysics-based translational research at the turn of the 19th century. Although debates about the actual composition of the cell membrane persisted over the years, Overton’s findings would end up standing the test of time. In 1976, Alan Finkelstein clarified these findings by carrying out a series of experiments studying the partitioning and diffusion of different solutes into nonpolar solvents. By studying solutes of different structures and sizes, he demonstrated that membrane permeability follows a coupled solubility–diffusion mechanism (Finkelstein, 1976).
The Meyer–Overton correlation. Anesthetic efficacy in a clinical setting correlates strongly with the partitioning ratio of a compound from the gas state into olive oil. Their findings suggested that cell membranes are also lipoid in nature. From Campagna et al. (2003), Fig. 3 is adapted with permission from the New England Journal of Medicine.
At the same time as the plasmolysis studies, the study of surface physics was developing (Roberts, 2013). Benjamin Franklin, in the late 1700s, was one of the first to describe the behavior of a drop of oil to spread out thinly on a body of water (Tanford, 2004). In 1890, Lord Rayleigh reported his investigations on contamination of water surfaces by olive oil and estimated that the thin film was formed by a lipid layer of 10–20 Å in thickness (Rayleigh, 1889). This work sparked the interest of Agnes Pockels, a young woman who spent her time at home, caring for her parents and the household. Although she did not formally attend school, her brother was a university student studying physics, and she was exposed to the literature and textbooks that he would bring home. In her daily kitchen work, she would observe the behavior of oil on water while she washed the pots and pans, and this made her wonder about the molecular structure and physical properties of these thin films. With clear passion and determination, she built an apparatus for measuring the surface tension of the thin oil films on water out of the kitchen pans that she was so familiar with. She contacted Lord Rayleigh, and in 1891, he helped her to publish her first independent paper and one of the first studies of surface tension (Pockels, 1892). Several decades later, Irving Langmuir adapted Pockels’s apparatus for higher-resolution measurements to build the Langmuir trough and with this solidified the conclusion that olive oil forms lipid monolayers on the surface that are 13 Å thick (Langmuir, 1917).
The development of the Langmuir trough provided the key equipment for high-resolution investigations of the structure of cell membranes. If membranes were formed by oil, as Overton’s experiments indicated, then they should behave similarly to the olive oil experiments performed by Lord Rayleigh, Pockels, and Langmuir. In 1925, Evert Gorter and Françoise Grendel extracted cell membranes from erythrocytes to measure the surface properties of the cell membrane using the recently developed physics approaches. The choice of erythrocytes was key to this experiment because these cells do not contain major organelles, and so the lipid fraction extracted would be expected to represent only the plasma membrane. They used the Langmuir trough to measure the surface area of the lipid molecules composing the cell membrane (Gorter and Grendel, 1925) and discovered that the area was exactly half of that expected if the molecules formed a monolayer. This led them to conclude that the cell membrane was a lipid bilayer, formed by two layers of oil molecules. This was the first proposal of such a structure and somewhat serendipitous considering their experimental approach was far from robust. Changes in the lipid extraction methods and surface pressure dependencies can alter the conclusions of the experiment (Bar et al., 1966), and so the debate between a monolayer, bilayer, or other structure remained for decades. Still, the seed of the idea of the cell membrane as a lipid bilayer structure was planted, which was a sufficient starting point.
In the same year, Hugo Fricke measured the electrical capacitance of intact erythrocytes in suspension (Fricke, 1925). With prior knowledge that the membrane was lipid impregnated, he used a reasonable assumption of the dielectric constant, ε = 3, and determined that the hydrophobic thickness of the cell membrane was 3.3 nm. In hindsight, this is a remarkably accurate measurement in agreement with higher-resolution methods used today like small-angle x-ray and neutron scattering (Kučerka et al., 2009). Perhaps more importantly, this experiment demonstrated that the cell membrane was an electrical barrier. Fricke was cautious not to overinterpret his results, but if we take the 10–20-Å monolayer thickness measured by Rayleigh, Pockels, and Langmuir, the data are in agreement with a lipid bilayer structure.
In 1935, James Frederic Danielli and Hugh Davson compiled the many results from Fricke, Gorter, and Grendel and others to develop the paucimolecular model (Danielli and Davson, 1935), i.e., a model with the minimum amount of necessary molecular detail. Technically, Fricke’s results allowed for membrane structures from unimolecular to trimolecular thickness, while Gorter and Grendel’s work suggested a bilayer with room for error. In the Danielli and Davson model, they propose a bilayer of ampipathic lipoid molecules filled with a nonpolar lipoid center ( Fig. 2 B ). On either side of the membrane is a protein layer at least one molecule thick adsorbed onto the lipoid surface because it was believed that proteins could not stably exist inside the membrane. In their model, it was these protein layers that were responsible for selective permeability, and they could form structures spanning the membrane to enable the passage of salts and sugars. This model consolidated many of the physical findings of the cell membrane, but the idea of the membrane structure as a lipid bilayer remained under heavy debate for many decades.
This changed in 1959, when J. David Robertson was studying the ultrastructure of myelin sheaths at the Nodes of Ranvier by electron microscopy. To improve resolution, he applied potassium permanganate (KMnO4) to stain structures, which highlighted a common trilaminar unit—two dark lines separated by a light center—that was observed at the plasma membrane boundary and encapsulating every organelle (Robertson, 1959). He interpreted the dark lines as the adsorbed protein layers and the light center as the lipoid, similar to the model of Danielli and Davson. The observation of the same structure being visible for all cellular compartments led him to propose the unit membrane model ( Fig. 2 C ), where he postulated that all membranes possess the same molecular structure. At that time, he even asserted that all membranes within a cell were in contact with one another, forming one continuous barrier. Although this idea is incorrect, the main point is that the lipid bilayer structure is something that is ubiquitous within biology, and it is capable of combining and fusing under the right conditions. In 1971, several studies measured the structure of membranes by x-ray diffraction (Levine and Wilkins, 1971; Wilkins et al., 1971), with results consistent with the lipid bilayer backing up Robertson’s imaging studies with high-resolution physical data. Finally, in 1972, Singer and Nicolson published their fluid mosaic model of the cell membrane ( Fig. 2 D ), which is essentially the model of the membrane that is used today (Singer and Nicolson, 1972). Although many students are introduced to this concept in a single figure in a biology textbook, it does not do justice to the comprehensive understanding of the membrane structure, membrane proteins, and the thermodynamics of the system as a whole that is presented in their paper.
The lipid bilayer is a completely conserved structure across all of biology. It provides a thermodynamically favorable solution to the physical problem of creating an electrical barrier. It is something that assembles spontaneously, driven by the hydrophobic effect, burying nonpolar acyl chains that are constrained by the polar and charged lipid head groups. The hydrophobic core provides an environment where water is rarely present, creating a low dielectric environment, where partial charges cannot rearrange to counteract the electrostatic field. Thus, the free energy required to transfer a charged particle from a high dielectric medium, like water, to the low dielectric of the membrane is highly unfavorable, making the leakage of ions prohibitive (Parsegian, 1969). Furthermore, this hydrophobic core is self-stabilized by interactions between the acyl chains of the phospholipid molecules. Thus, it is a barrier that possesses structural integrity but also the properties of elasticity and pliability that have been described. With the lipid bilayer, a cell builds a self-assembling, self-repairing, practically impermeable barrier for charged particles and ions, enabling the storage of information and potential energy to fuel biology’s work. It provides a natural and comprehensive solution, basically for free, and lets biology focus on the harder problem of enabling selective permeability.
A new class of protein that resides in membranesPlasmolysis studies also paved the way toward understanding the species responsible for selective permeability. Two of the pioneers in this field of work were Jacques Loeb and W.J.V. Osterhout, the first two editors of JGP. They independently performed some of the first studies of cell permeability in the presence of inorganic salts (Osterhout, 1911; Loeb, 1912). In the first of these studies, Osterhout repeated Overton’s experiments of plasmolysis in Spirogyra but this time studied the cells over a longer period of time. When placing the cells in a concentrated salt solution, he observed Overton’s initial result that the protoplast shrank away from the cell wall. However, over a longer time period, he observed the protoplast returned to its original shape and volume, which meant that the membrane was actually permeable to these inorganic ions. He went on to conclude that this must mean that the membrane was not composed of lipoid as previously asserted but instead was proteoid in nature. Perhaps this was too strong a conclusion given prior results, yet it highlights several truths about permeability across cell membranes. First, the passage of charges across the membrane must be electrostatically supported by protein, and second, membranes actually contain a large fraction of protein. A comprehensive review of permeability, and more on the work of Loeb and Osterhout, can be found in a recent Milestone article in JGP (Hille, 2018). In the following sections, I highlight a few key studies that led to the solidification of integral membrane proteins as a new class of hydrophobic proteins that are responsible for the proteoid nature of cell membranes.
The earliest ideas of proteins existing inside the cell membrane came from deductions based on physiological observations. In many cell types studied, Na + was observed to be at a higher concentration outside of the cell, while K + was found to be higher inside. In 1941, R.B. Dean proposed that to maintain these physiological concentrations, there must be a pump responsible for actively moving these ions against their gradients and that it may reside within the cell membrane (Dean, 1941). In 1957, Peter Mitchell supported this idea, proposing the existence of translocases, enzymes that catalyze substrate transport across the membrane (Mitchell, 1957), and introduced models of integral translocases with alternating access transport mechanisms. The discovery of the Na + /K + ATPase by Jens Skou in 1957 (Skou, 1998) identified a specific membrane protein responsible for active transport; however, it still remained unclear as to how this protein was associated with the cell membrane. At that time, both the Davson–Danielli and Robertson membrane models did not account for protein inside the lipoid core as polar and charged protein molecules were expected to destabilize the membrane structure. Further evidence would be required before it would become accepted that proteins could span the hydrophobic membrane.
A key development along the path to identifying integral membrane proteins was the discovery that one could purify fragments of membrane and reconstitute their electrical permeability behavior. In 1962, the first BLM ( Fig. 4 A ) experiments were conducted, in which a membrane was reconstituted outside of any cellular structure while maintaining similar electrical properties to intact cell preparations (Mueller et al., 1962). Higher-resolution BLM experiments would later reveal discrete, step-like addition of currents that contribute to the overall electrical permeability (Bean et al., 1969) indicating that whatever was responsible for the permeability, the behavior could be resolved in a quantal manner. Finally, in 1970, the first single-channel recording was measured by adding the antibiotic peptide Gramicidin to a lipid bilayer (Hladky and Haydon, 1970). Gramicidin showed conductance behavior that was step-like, with constant step magnitudes ( Fig. 4 B ). With this, permeability could be attributed to the peptide itself, and the macroscopic behavior dissected into a sum of individual channel conductances in the membrane. This led to the idea of proteins in the membranes facilitating membrane transport by creating “permanent or transient modifications of the bilayer structure.”
Electrical reconstitution of membrane permeability. (A) The BLM preparation for electrical measurements of permeability, adapted from Tien (1968). (B) The first single-channel recording of Gramicidin in lipid bilayers. From Hladky and Haydon (1970), Fig. 4 is adapted with permission from Nature.
However, this was not enough to deduce that the protein was spanning the membrane. For example, Gramicidin could be acting as a rapid carrier, shuttling ions across the lipid bilayer, as is the case for ionophores like valinomycin. Alternatively, the protein could be modifying the membrane in some defined way that facilitates the passage of ions, akin to the ideas presented in the past, e.g., the Davson–Danielli model. To get to the idea of actual integral membrane proteins with defined structures, parallel efforts in membrane protein biochemistry were pivotal. It was long known that biological cell membranes were composed of proteins in high amounts; however, purification of these proteins led to an interesting result. Proteins obtained from the membrane fractions were mainly insoluble in water and often aggregated (Richardson et al., 1963). This suggested that the proteins that associated with membranes were somehow different in their physical nature compared with the vast number of soluble proteins studied so far. The next big piece of evidence came from electron microscopy freeze-fracture studies that split the lipid bilayer between the two leaflets. This directly showed that there were defects in the membrane that would appear as a positive signal in one leaflet, while leaving a negative impression, or hole, in the opposite layer (Moor and Mühlethaler, 1963). This provided the necessary evidence that proteins associated with membranes were in fact embedded in the lipid bilayer. With all of this accumulating evidence, Singer and Nicolson proposed the mosaic part of their fluid mosaic model (Singer and Nicolson, 1972), the idea of integral membrane proteins that are hydrophobic in nature and reside stably within the hydrophobic lipid bilayer. With further research in purification of membrane fractions and functional reconstitution (Miller and Racker, 1979), the idea of integral membrane proteins acting as ion channels, transporters, active pumps, and receptors soon became accepted.
As was the case with Robertson’s electron microscopy studies, visual evidence is often required to remove remaining doubts. Fortunately, for the study of integral membrane proteins, there was a serendipitous source of highly ordered proteins in membranes that could be reasonably obtained from a type of archaea, Halobacterium halobium. These halobacteria were discovered to contain large, visible patches of purple membranes (Stoeckenius and Rowen, 1967). Dieter Osterhelt and Walter Stoeckenius developed methods for isolating these membrane fragments that contained nearly pure amounts of the protein bacteriorhodopsin (Oesterhelt and Stoeckenius, 1971). These preparations resulted in protein that was already in ordered two-dimensional crystals within the membrane, and with this, in 1975, Richard Henderson and Nigel Unwin reported the first structure of an integral membrane protein of bacteriorhodopsin at 7 Å (Henderson and Unwin, 1975). It would take a decade before an atomic resolution structure of another membrane protein would be determined, with the delay being attributed to a fundamental biochemical challenge of membrane proteins. Protein crystallography requires the formation of highly ordered three-dimensional crystals from protein in solution. How, then, can membrane proteins be made soluble when they are hydrophobic and prone to aggregation? The solution came via the use of detergents and micelles as membrane mimetics (Helenius and Simons, 1975; Tanford and Reynolds, 1976). Detergent has the capability of extracting protein from the membrane, replacing the surrounding lipid, and, in some cases, supporting the membrane protein in a folded and functional state. With this key development, in 1985, Johann Deisenhofer, Harmut Michel, and others reported the first atomic-resolution, 3.0-Å crystal structure of the photosynthetic reaction center of Rhodopseudomonas viridis (Deisenhofer et al., 1985). With the ability to handle membrane proteins in solution, biochemical optimizations became tractable, and along with that came more structural information. In 1991, the structure of the bacterial outer membrane porin OmpF was solved at a remarkable 1.8-Å resolution (Weiss et al., 1991), on par with the resolution being obtained for soluble protein structures. In 1998, Rod MacKinnon and his laboratory determined the first structure of a potassium ion channel, revealing the elegant pore structure conferring selective K + permeability (Doyle et al., 1998). Since then, many structures of channels, transporters, pumps, and receptors have been solved, and today, they arrive at a steady pace, especially with recent technological developments in cryo-electron microscopy and single-particle reconstruction (Hendrickson, 2016). Although resolution is often lower for membrane proteins compared with soluble proteins, it is not an absolute fact, because many structures of membrane proteins have improved in resolution over time. One remarkable example is the structure of the yeast Aquaporin Aqy1 solved to a sub-angstrom 0.88-Å resolution (Eriksson et al., 2013). Still, it remains true that our knowledge of the structures of membrane proteins, and particularly eukaryotic membrane proteins, falls well behind that of soluble proteins and that this is a consequence of the inherent biochemical challenges associated with expressing and purifying stable membrane proteins.
The physical behavior of membrane proteins in membranesAs stated by Singer and Nicolson, the membrane is a “two-dimensional liquid-like solution of monomeric or aggregated integral proteins … dissolved in the lipid bilayer” (Singer and Nicolson, 1972). Yet, the membrane solvent is much more complicated than the other biological solvent that we are used to thinking about, i.e., water. The lipid bilayer has a defined structure and along with it has macroscopic material properties. The chemical composition of membranes is also extremely diverse. In the past this has been interpreted as lipids playing a passive role in membrane protein reactions, merely providing the sea of solvent in which the protein floats about. Following this, many studies of membrane protein biochemistry do not involve lipid bilayers but instead study the protein in detergent micelles, a practical necessity for purification in the aqueous state. This approach is well justified if the protein’s function is maintained in a micellar environment, which can be the case when it is dominated by the intrinsic protein structure. However, it cannot be denied that standard methods of membrane protein purification strip away the natural solvent, placing the protein into an environment that could miss essential chemical or physical properties of the original lipid bilayer. Therefore, the reactions of membrane proteins, whether it is ligand binding, conformational stability, folding, or oligomerization, all depend on the free energy of the system as a whole, and this means in the context of the lipid bilayer.
Although the lipid bilayer structure is generally constrained to the 3-nm hydrophobic core measured by Fricke, it exists across biology with practically infinite chemical compositions that vary molecular head group and acyl chain chemistry. This diversity appears to be a result of an organism’s need to maintain an electrical and physical barrier while preserving membrane fluidity under changing environmental conditions (Siliakus et al., 2017). Along with this diversity comes an apparent ability for specific lipids to regulate membrane protein function. Early studies of membrane protein reconstitution found that not all lipid bilayers were equivalent in terms of protein function and stability (Miller and Racker, 1979). As a result, many studies emphasized the use of lipid compositions as close to native conditions as possible. Furthermore, specific lipids were identified as essential for the proper gating activity, most notably the phosphoinositides (PIP2; more on this particular lipid as a signaling molecule is described in the recent JGP Milestone review [Hilgemann et al., 2018]). Simply put, function, or lack thereof, is the strongest piece of evidence that particularities in lipid chemical species are important for the biological role of these proteins.
From a structural perspective, many high-resolution crystal structures of membrane proteins have resolved specific lipids directly associated with the protein. In some of these, the lipids were added during purification, and they became ordered during crystallization. However, in other examples, the lipids appear to have come along for the ride, remaining bound even in the harsh conditions of detergent extraction. One example is the K + channel KcsA, which was observed to bind a single negatively charged lipid per subunit ( Fig. 5 ), and this lipid appears to be necessary for function (Valiyaveetil et al., 2002). High-resolution electron crystallography structures of native crystals of Aquaporin-0 (Aqp0) show the proteins are organized in a lattice, stitched together by a layer of lipids ( Fig. 5 ), with a high degree of specificity for certain lipids at different sites (Gonen et al., 2005; Hite et al., 2010). Recently, mass spectrometry analysis has provided more quantitative information about lipid binding and has been able to describe the specificity for interactions over other lipids (Laganowsky et al., 2014), as well as the ability for lipids to shift oligomeric distributions in detergent micelles (Gupta et al., 2017). Are these lipids that are found to interact with membrane proteins in structures or mass spectrometry analogous to crystallographic waters found in soluble proteins? Or do they represent something more like ligand binding in the context of the membrane? Electron spin resonance and fluorescence-quenching studies do not support the idea of long-lived lipids binding to membrane proteins in membranes (Lee, 2003). However, it is likely that further research is required with the new structural data in hand to provide context of the reactions in the membrane.
