1.1. Why Study Membranes and Membrane Proteins?
Biological membranes and membrane proteins, responsible for numerous exciting biological processes, present one of the paramount challenges in biophysics today. Membranes are present in great number and variety in all organisms. They form the boundary between the inside and outside for any bacterium or cell, and they delimit the host of organelles that make up their inner subunits. Each biological membrane is made up of dozens of different types of lipids and sterols, and any particular type of membrane has a characteristic content of these different constituents. As a very basic example, we mention that prokaryotic membranes contain a notable component of negatively charged lipids but almost no cholesterol, while eukaryotic membranes are mostly zwitterionic but have a significant amount of cholesterol. Since the driving biophysical principles of membrane formation are very simple—they lie in the amphipathic properties of any lipid molecule—a single lipid type is sufficient to form membrane-like bilayers in an aqueous environment. Such model membranes are used extensively to study biophysical properties that are representative for most membrane systems. A particularly interesting effect is observed when detergent molecules are added to lipid bilayer samples: the detergents solubilize the bilayers, and in certain regimes so-called bilayered mixed micelles or “bicelles” are formed. In the simplest case, they can be described as microscopic disks where a bilayer patch is encircled by a “rim” of detergent molecules. Bicelles represent a new instance of lipid morphology and are extensively applicable to structural studies of lipid membranes and protein structure.1
Membranes delimit any cell and all of its compartments. They form natural borders for metabolic substances and signaling molecules. Membrane proteins are the porters and gatekeepers that make sure that only proper molecules or signals make it across the membrane. Since membrane proteins perform numerous key functions in cell metabolism and signaling, they contribute over 30% of the genes in typical eukaryotic genomes,2 and they form the targets for over 50% of drugs in use today.3 The number of elucidated structures of membrane proteins has grown exponentially after the first structure was published in 1985, thus equaling the rate at which structure determination of soluble proteins emerged early on.4 Still, the number of available high-resolution structures of membrane proteins is limited. There are Internet sites that keep track of newly published structures of membrane proteins. The crystallography-oriented Web site of Dr. Stephen White [http://blanco.biomol.uci.edu/mpstruc] has recently been joined by another site maintained by Dr. Dror Warschawski that is dedicated to structures of membrane proteins elucidated by nuclear magnetic resonance (NMR) spectroscopy [www.drorlist.com/nmr/MPNMR.html]. Another equally important site of Dr. Hartmut Michel [www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html] with an emphasis on crystallization conditions is no longer updated, but states that access is still enabled.
In this review article, we aim to give a general overview of lipid bicelles as employed in the study of protein structure. Recent advances in the field of protein structural biology that have been made possible by exploiting the unique properties of lipid bicelles, in both solution and solid-state NMR spectroscopy, will be discussed. During the last five years, review contributions have presented bicelles either within the far more general context of reconstitution media for solution NMR studies (see section 1.4) or have focused on macroscopically aligned bicelles as used for solid-state NMR studies.5,6 One very recent contribution has tackled the formidable task of reviewing all membrane mimetics employed in both solution and solid-state NMR studies.7 As mentioned above, we will limit the contents of this review article to applications of lipid bicelles, but will cover both the isotropic and the aligned bicelles as used in NMR studies. Some parts of this article can be viewed as an update on the review articles of Opella and Marassi,8 Marcotte and Auger,9 and Prosser et al.10 In addition, some of our own recent research involving bicelles is presented in detail.
1.2. Understanding Atomistic-Level Structures Is Important
The intrinsic properties of a cell membrane originate from interactions among molecules like amphipathic lipids, polysaccharides, cholesterol, proteins, and water. Since the chemical and physical properties of these molecules differ considerably, the minimum free energy of mixing corresponds to a heterogeneous cell membrane. Domains rich in protein, cholesterol and anionic lipids, and rafts have been reported to play important roles in biological activities of cells which have direct implications in viral infection, bacterial infection, amyloid toxicity related to aging diseases, and cancer.11−13 For example, the presence of charged lipids in bacterial cell membranes and their absence in mammalian cell membranes are one of the key factors in the selectivity of antimicrobial peptides. Likewise, cholesterol present in mammalian and absent in bacterial cell membranes has been shown to have a similar influence on the selectivity of antimicrobial peptides.14 In addition, the process of folding, misfolding or refolding, and aggregation of amyloidogenic proteins in cell membranes is different from that in solution, and also depends on the composition of the cell membrane.15 Needless to mention that the secondary and tertiary structures of proteins can be different when they associate with the cell membrane. Therefore, high-resolution structure of individual molecules and their orientation in a membrane environment could reveal the factors that drive the molecular association and their function in this heterogeneous membrane environment. While solving the atomic-level structure of a membrane protein still remains a big challenge for most biophysical techniques, the increasing number of structures determined by X-ray and NMR studies continue to shed light on the functional aspects of membrane proteins. For example, the reported high-resolution structure of the potassium channel forming membrane protein16−18 has provided insights into the geometry of the channel, ion selectivity, interactions between lipids and the protein, and the role of individual amino acids in the transportation of potassium ions.
1.3. NMR Is an Ideal Technique to Measure Structure and Dynamics
NMR spectroscopy has played a pivotal role in the structure determination of a host of biomacromolecules, ranging from proteins to nucleic acids. Importantly, NMR spectroscopy has provided scientists with detailed structural and dynamical information that is inaccessible through other biophysical means. First and foremost, X-ray crystallography has elucidated a tremendous number of protein structures in high resolution. The environment of a protein crystal, however, is far from physiological and may shadow important aspects, especially of protein dynamics. In this respect, NMR spectroscopy is both an alternative as well as a complement to X-ray crystallography. The branch of NMR spectroscopy that deals with molecules in solution is known as solution-state NMR spectroscopy. It offers varied, well-tested, and sophisticated tools,19−25 to routinely deal with any soluble protein that does not exceed a certain molecular weight. The upper limit for molecular weight is currently around 100 kDa26 and is continually pushed higher. Lipid membranes are typically not amenable to be studied by solution-state NMR spectroscopy, since they are well above the molecular weight limit. It is often possible, though, to study the structure of membrane proteins when they are solubilized by properly chosen detergents.27 Membrane proteins are notoriously hard to study since their highly hydrophobic nature routinely causes misfolding and aggregation, making it very hard to crystallize them in sufficient quality for X-ray diffraction.28 In addition, their slow reorientation in a membrane environment prohibits the use of well-established solution-state NMR methodology. The branch of solid-state NMR spectroscopy is rapidly evolving to deal with membrane proteins that are beyond the size limit for solution-state NMR spectroscopy.
Since the NMR observables chemical shift anisotropy and dipolar coupling are sensitive to both the chemical environment and molecular motions, they can be used to probe molecular structure and dynamics associated with biological processes such as ligand binding, conformational exchange and protein–protein interactions. One of the unique advantages of NMR spectroscopy is its ability to interrogate molecular dynamics over a wide range of time scales. Through NMR, motions from nanosecond to microsecond time scales can be probed via measuring different NMR parameters such as spin–lattice relaxation (T1), spin–spin relaxation (T2), relaxation in the rotating frame (T1ρ), residual dipolar couplings, and quadrupolar coupling (for nuclei with spin > 1/2). Thus, NMR spectroscopy is able to paint a very detailed picture of a system, where structure and dynamics as well as function can be correlated. Membrane proteins exhibit a broad time scale of dynamics and these motions highly influence the function of the protein: The residues in transmembrane segments generally undergo restricted motion on a fast time scale (picosecond-nanosecond), while soluble domains show large amplitude motions with slower correlation times. Loop regions move with intermediate amplitudes on intermediate time scales since they are anchored at transmembrane segments. The entirety of domains may perform collective motions like conformational changes at very slow time scales (microsecond). Typical dynamic properties of different regions were quantified on bacteriorhodopsin by extensive 13C NMR studies.29,30 Therefore, NMR techniques are well suited to study the dynamical structures of membrane proteins. Another unique advantage of NMR spectroscopy is that it can determine the orientation of a membrane protein relative to the lipid bilayer.
In the context of NMR studies of membrane proteins, lipid bicelles have opened completely new ways of preparing samples for NMR studies. This is mostly because the size of lipid bicelles can be custom-tailored for specific tasks. An additional unique property of certain bicelle preparations is their propensity to macroscopically align when brought into an external magnetic field. As a consequence, bicelles disobey the traditional classification of NMR experiments and notoriously cross the border between solution-state and solid-state NMR spectroscopy. Figure Figure11 gives a graphic overview of the position of lipid bicelles in NMR studies of membrane proteins. Care must be taken to prepare a well-behaved sample for successful structural studies using NMR spectroscopy. As is the case in the study of any membrane protein, the protein needs to be supplied in sufficient amount and purity, needs to have a specific isotope labeling scheme, and needs to be properly folded and reconstituted. Only then can it be taken into formulations that are suitable for NMR spectroscopy. Typically, those have been detergent micelles for solution NMR studies, and multilamellar vesicles (MLVs) of lipid for solid-state NMR studies. Lipid bicelles open a middle ground between these two model membranes, namely, micelles and MLVs. Since their size can be chosen to be small enough to tumble quickly on the NMR time scale, small bicelles (also known as isotropic bicelles) can be investigated using solution NMR experiments. Larger bicelles, especially when aligned macroscopically, are amenable to static solid-state NMR spectroscopy. In addition, magic angle spinning (MAS) NMR experiments can be applied to lipid bicelles.
1.4. Need for Excellent Model Membranes
The overall architecture of membrane proteins shows little variation: integral membrane proteins transverse the lipid bilayer of the cell membrane either as a single α-helix, or as a bundle of α-helices, or they form β-barrels. Since the differences in membrane protein architecture responsible for a specific function are often subtle, excellent model membrane systems are needed. In addition, the secondary and tertiary structures, folding, aggregation, dynamics, stability, orientation, and function of a membrane protein highly depend on the nature of the membrane environment. This is true even if membrane proteins are intrinsically tolerant to changes in the composition of the surrounding membrane.31 For example, the choice of a good detergent system was found crucial in studies of the enzyme PagP, an integral membrane protein forming a β-barrel. The detergent used initially was found to deactivate the enzyme because its structure is too similar to the substrate. Only with a more distinct detergent could an active enzyme be studied.32 Likewise, specific polyunsaturated side chains are present at high molar ratios in the lipids of rod outer segment disk membranes and accumulate near rhodopsin, an integral α-helical membrane protein.33,34 In the case of the antimicrobial peptide gramicidin A, suitable conditions had to be established to distinguish the physiologically relevant conformation from other conformations.35,36 The general awareness of the distinction between physiologically relevant and other conformations has obviously faded recently and had to be called back to mind.37
Different types of model membranes have been used for NMR studies. The use of TFE/water mixtures is no longer considered to be a good model membrane. Detergent micelles and lipid vesicles have commonly been used in solution and solid-state NMR applications, respectively. While the use of micelles enables the applications of well-established solution NMR techniques, the potential impact of the curvature of micelles on the structural folding remains a concern. Therefore, a planar lipid bilayer is considered to be a better model membrane than a micelle. As mentioned earlier, bicelles that are devoid of acute curvature like a micelle are considered to be a more suitable model membrane for NMR studies. Nevertheless, micelles have been found to be useful in trapping transiently lived helical structures of amyloid proteins that otherwise rapidly convert into β-sheet structures in a lipid bilayer.38,39
The importance of detergents in the study of solubilized membrane proteins has been reviewed,40 at times under imaginative titles referring to detergents as “French swimwear”41 or denying that they are part of a soap opera.42 In view of the advantageous properties of bicelles over detergent micelles, another review title states that “small is beautiful, but sometimes bigger is better.”43 Two other review contributions have reported on bicelles in the context of membrane mimetics and solubilizing agents for solution NMR spectroscopy.26,44 These reviews cover micelle-forming detergents as well as innovative solubilizing approaches other than bicelles, such as in situ NMR,45 amphipols,46 or nanodisks47,48 which are not within the scope of the current review. A comparison of NMR spectra acquired on different membrane proteins in bicelles and nanodisks, both isotropic and aligned, has been performed.49 Bicelles were investigated as novel surfactants in the context of cell-free expression of membrane proteins.50 Cell-free production of integral membrane proteins in bicelles was compared to production in lipid protein nanodisks as well as micelles and liposomes.51 Subunits a and c of ATP-synthase have been produced by cell-free synthesis in the presence of bicelles; subunit a was shown to have a similar fold to native protein extracted from bacterial cell walls.52