Introduction to the Structural Biology of Membrane Proteins

MARY LUCKEY

1.1 Introduction

The biomembrane is an essential feature of life, defining the interior of cells and controlling the entry and exit of molecules and ions. Lipids in the membrane make a hydrophobic barrier for aqueous solutes; this barrier is semipermeable due to the presence of membrane proteins. In addition to transport functions, membrane proteins have roles as receptors, enzymes, and structural components. Biomembranes also contain glycolipids and glycoproteins, whose carbohydrate constituents are external to the cell and are important in recognition. This introduction focuses on membrane proteins in the context of their lipid environment.

Membrane proteins share some features with soluble proteins, for example the protein interior (excluding channel walls) is tightly packed with mostly nonpolar residues. However, the surface of membrane proteins is amphiphilic, allowing interactions with both the polar exterior and the nonpolar interior of the membrane. The need to maintain this amphiphilic structure increases the difficulty of purifying and handling membrane proteins. In addition, their localization in the membrane often impedes their overexpression. These difficulties hampered their structural characterization for many years, so that X-ray crystallography of membrane proteins lagged 25 years behind that of soluble proteins. In 1988, Harmut Michel, Johann Deisenhoffer, and Robert Huber received a Nobel Prize for the first crystal structures of membrane proteins, those from the photosynthetic reaction center of Rhodopseudomonas viridis. (Rhodospeudomas viridis has been renamed Blastochloris viridis.) Over the next two decades, new membrane protein crystal structures were sufficiently rare to grace the covers of Nature and Science when they did appear. However, their number rose exponentially; the data base now contains more than 650 unique structures of membrane proteins determined with X-ray crystallography or NMR spectroscopy, with good overall agreement in structures that have been solved by both. The availability of detailed membrane protein structures allows researchers to further probe their function and dynamics using genetic, biochemical, biophysical, and computational tools.

1.2 Membrane Features

The native environment of membrane proteins is dynamic and asymmetric, described by Singer and Nicolson as fluid and mosaic in their 1972 model that became the paradigm for membrane structure (Figure 1.1). Lipids consisting of polar head groups and nonpolar acyl chains form a two-dimensional fluid bilayer (Figure 1.2). Lipid composition is diverse, with most of the lipids randomly distributed in the bulk phase of the bilayer while some are localized by specific interactions with proteins and/or other lipids, often in regions of ordered lipids called rafts (see below).

In the lipid disordered phase (Ld, also called La), fluidity of the bilayer results from the constant and varied motion of lipids (Figure 1.3). Although the lateral diffusion rate in pure lipid bilayers is very fast, the measured mobility of bulk lipids on the surface of cells is much slower. This difference has been explained by single-particle tracking on the surface of cells containing cytoskeletons: single-molecule trajectories are fast within small regions, where they are confined until they hop to a contiguous region, producing a slower overall progression.

The mosaic distribution of membrane proteins results from wide variations in lateral mobility, from those that diffuse rapidly on the surface to those anchored by the cytoskeleton. A large proportion of membrane proteins function in protein assemblies, which themselves have varying lifetimes. Some complexes of membrane proteins are very stable, such as the respiratory complexes involved in energy transduction (see below), while others are the result of transient interactions such as those involved in signal transduction. Many lipid-anchored proteins are observed in membrane rafts enriched in sphingomyelin and cholesterol in the lipid-ordered (Lo) state (see Figure 1.2). The presence of rafts varying in size (diameters from 10 to 200 nm) and in duration (from <1 ms to fairly stable lifetimes) enhances nonrandom distributions and dynamic interactions in the membrane. Lipid-lipid interactions probably drive raft formation, given that even simple lipid mixtures reveal fluid immiscibility (Figure 1.4). The fused hydrocarbon rings of cholesterol are nearly rigid, allowing the sterol to align with lipids containing saturated acyl chains, especially sphingomyelins, and promote the tight packing of the Lo phase.

1.3 Lipid Polymorphism

Of the possible physical states for lipid aggregates, only a few are present at biological conditions, while others are useful in the laboratory. A pure lipid bilayer (constituted by only one lipid species) can be in lamellar crystalline (Lc), lamellar gel (Lß), or lamellar liquid crystalline (called fluid, La, or disordered, Ld) states, depending on the temperature and pressure. The shape of the lipid molecule may introduce significant curvature to the bilayer, an important force during some membrane processes. Since excessive curvature disrupts the bilayer, biological membranes contain a significant fraction of non-bilayer lipids (lipids such as phosphatidylethanolamine that do not form bilayers in the pure state) to minimize the curvature tension. Non-bilayer states include hexagonal phases (hexagonally packed arrays in long cylinders) and cubic phases (cubic packing of rod-like elements, Figure 1.5). Hexagonal phases may be important in biological transitions, such as pore formation and membrane fusion. The lipidic cubic phase (LCP) has become an important tool in the crystallization of membrane proteins.

In contrast to the simplified mixtures employed for physical studies, the lipid composition of most membranes is very complex: a typical membrane has about a hundred lipid species, and over 1000 different species of lipids have been identified in biological samples. Prokaryotic organisms vary the fatty acid content of their membranes to maintain fluidity at extreme temperatures (longer and more saturated fatty acids have higher melting points than shorter and unsaturated fatty acids). Eukaryotic organisms have markedly different...