The side chains are blank______ among amino acids.

5 Conclusions

IMP disorder has been formally hypothesized15 now for over 5 years with many earlier reports of membrane protein dynamics16 open to reinterpretation in such a context. With the premise and existence of disorder within extracellular and cytoplasmic domains of TM proteins further conceptually developed,21 the scene was clearly set for new two-way evaluations of disorder and mechanisms TM proteins utilize to perform their function, including clustering, trafficking, and the inter-relationship of PTM and protein conformation. The single-pass IMPs emphasized here in the context of TM protein disorder highlight the probable impact of regional flexibility in normal physiological function of IMP as well as challenges and possible insight into pharmacological control of regional disorder to modulate IMP function in various disease states. Incorporation of disordered regions into essential cell surface receptors likely enhances formation of functional networks necessary for adaptable and efficient cross-membrane signal transduction. Nevertheless, there is much yet to be deciphered as to the consequences of intrinsic disorder for the conformation and in turn function of IMP with unstructured domains. This includes the specific assignment of presumed increased functionality to regions of disorder as well as the physiological effects of the order-disorder (and vice versa) transitions possibly occurring due to interaction with natural ligands or to pathological mutations. As highlighted in this chapter for a few TM protein examples, some separate facets of this have been done for individual proteins. It is clear that an integrative approach of bioinformatics, biophysical assessments, in vitro assembly models, and targeted mutagenesis will be the vital key for future successes in the field.

Type I and Type II membrane proteins

Integral membrane proteins may be further subdivided. Many integral membrane proteins possess a single transmembrane sequence. These may be divided into type I membrane proteins, which have a cleavable N-terminal signal sequence and a transmembrane sequence that is usually situated close to the C terminus. Type II membrane proteins have a noncleavable hydrophobic transmembrane region close to the N terminus, which serves as a combined signal/anchor sequence. Examples of type I membrane proteins include the histocompatibility antigens, glycophorin and membrane immunoglobulin. Examples of type II membrane proteins include the transferrin receptor, the asialoglycoprotein receptor, and many ecto-enzymes and glycosyl transferases.

Many integral membrane proteins span the membrane more than once, and often many times. Either terminus may be inside or outside the cell. Proteins with multiple transmembrane domains include a large family of G-protein-coupled receptors such as rhodopsin, the coloured visual pigments, and receptors for many small molecules, as well as many pumps and channels.

Abstract

Integral membrane proteins adopt diverse structures with differing stability, flexibility, and oligomeric state. How much of this is dictated by the amino acid sequence and how much by the membrane is unknown, as are the key features that have to be mimicked in vitro to stabilize a functional membrane protein fold. Here we summarize successful approaches to fold helical membrane proteins and outline advances in kinetic studies in vitro. We also describe how studies are progressing to more complex, larger, and multisubunit proteins and put the work into context with regard to the insertion machinery involved in vivo.

2 Integral Membrane Proteins

Integral membrane proteins penetrate the lipid bilayer. These glycoproteins express carbohydrate residues on the outside surface of the cell. They contribute negative charge to the cell surface, function as receptors or transport proteins, and carry RBC antigens (Chasis and Mohandas, 1992; Mohandas and Chasis, 1993; Schrier, 1985). Band 3 (anion exchanger 1) is the major integral protein. It accounts for approximately one-fourth of the total membrane protein, with about 106 copies/RBC (Delaunay, 2007; Schrier, 1985). It is important as an anion transporter and provides a site for binding of the cytoskeleton internally. Additional transmembrane glycoproteins called glycophorins also help anchor and stabilize the cytoskeleton (Chasis and Mohandas, 1992).

Membrane Components

The membrane lipids include phospholipids, sphingolipids, and cholesterol (see Chapter 11).

The phospholipids contain two fatty acids (usually 16 to 18 carbons) attached to glycerol in addition to a phosphate group. The fatty acids may be either unsaturated or saturated. Most phospholipids have ethanolamine, choline, inositol, or serine esterified to the phosphate.

The sphingolipids include sphingomyelin, cerebrosides, and gangliosides. The cerebrosides and gangliosides, sugar-containing lipids called glycosphingolipids, are located primarily in the plasma membrane. Sphingomyelin is prominent in myelin sheaths.

Cholesterol is primarily found in the plasma membrane with its hydroxyl group on the surface at the water interface.

Membranes are generally 40% to 50% protein but can range from extremes such as 20% protein in the myelin membrane to 80% protein in the inner mitochondrial membrane. Protein and lipid composition is unique for each membrane, and their distribution is asymmetric.

Elimination of Misfolded Proteins From the Endoplasmic Reticulum

Integral membrane proteins and secretory proteins fold and assemble in the lipid bilayer or lumen of the ER (see Fig. 20.8). Proteins that fail to fold or assemble are retrieved from the ER and degraded by the proteasome in a pathway known as ERAD (ER-associated degradation). The ERAD pathway also regulates levels of a number of ER resident proteins. ERAD target proteins are detected either by a chaperone in the ER lumen, or directly by a large multi-protein complex inserted in the ER membrane. Either way, the substrate is retro-translocated by that complex back to the cytoplasmic surface of the ER where it either has its trans-membrane domains cleaved in the plane of the membrane by specific proteases or is captured, forcibly extracted from the membrane by an AAA-ATPase and ubiquitylated by one of two dedicated E3 ligases prior to degradation by proteasomes.

Medical interest in the ERAD pathway arises because defects in ubiquitylation of particular proteins are associated with the pathology of Parkinson disease. Furthermore, the most common form of cystic fibrosis results from ERAD-mediated degradation of a slow-folding (but catalytically competent) variant of the CFTR (cystic fibrosis transmembrane regulator) ABC (adenosine triphosphate binding cassette) transporter (see Fig. 17.4) before it can be exported to the cell surface.

3.08.1.1 Classes of Membrane Proteins

Integral membrane proteins account for approximately 30% of the human genome but represent almost 50% of the targets of pharmaceutical agents.6,7 Compounds modulating ion channels, transporters, or G protein-coupled receptors (GPCRs) have routinely been among the most widely prescribed therapies for several decades. There are multiple reasons for this, including not only the involvement of membrane proteins in many disease-related regulatory processes but also their inherent druggability arising from binding sites which have evolved to recognize small molecules.

Despite their popularity as drug targets, membrane proteins have consistently been poorly represented in collections of X-ray structures of proteins, primarily due to difficulties in purifying sufficient quantities of high-quality protein stable enough to form regular crystals. Examples were until relatively recently limited to a handful of heroic efforts, such as the photosystem complex.8 However, between 1998 and 2002, breakthrough structures (described below) were achieved for ion channels, transporters, and GPCRs. A consistent theme for each of these classes of proteins has been the focus on particular family members, which, while still challenging, yielded valuable information before progressing to more difficult examples.

In this section, we will introduce the main classes of membrane proteins, which have been the focus of structural biology efforts. It is not possible to give a comprehensive analysis of the field in a single article; instead, we give an outline of the progress for each major target families here.

Membrane Proteins

Integral membrane proteins present special problems because of their location within membranes and because they are not soluble in aqueous buffer solutions. The first step will be to obtain a preparation of the membrane of interest, usually by differential centrifugation. Next, the protein has to be extracted from the membrane preparation, most commonly by using solutions of detergents such as Triton X-100, Lubrol PX, digitonin, sodium cholate, etc. This is a crucial step and the best detergent to use to obtain optimum release of the protein from the membrane fragments can be determined only by trial and error.

Once a soluble extract of the protein has been obtained its purification can be achieved using the usual chromatographic techniques except that, because of solubility problems, it will be necessary to maintain a standing concentration of detergent in the buffers. This frequently adversely affects the performance of ion exchange materials and more success in isolation of membrane proteins has been achieved by exploiting their binding properties, that is, by using various forms of affinity chromatography.

A final problem, once the protein has been purified, will usually be to remove the detergent from the preparation or to change the detergent type. This can be achieved by a variety of methods, including equilibrium dialysis, gel filtration and a variety of chromatographic methods.

Peripheral membrane proteins, that is, those that are only loosely associated with the membrane, do not usually present special problems. They can be released from membrane preparations by salt extraction or by changes in pH, are usually soluble in aqueous buffers, and are amenable to the usual purification methods.

6.4.1 Back to First Principles

Integral membrane proteins have at least one transmembrane domain that crosses the lipid bilayer. These transmembrane (TM) domains are naturally enriched in apolar amino acids that allow a smooth insertion in the apolar phase of the lipid bilayer. Basically, a TM domain consists in a cluster of ∼25 apolar amino acid residues with a α-helical structure. Classifying the amino acids according to their hydropathy had allowed Kyte and Doolittle to propose a hydropathy/hydrophobicity scale25 that has been widely used as an algorithm for the prediction of membrane protein topology.26 However, the rapid progress of bioinformatics approaches has rapidly supplanted this early approach by machine learning methods that extract statistical sequence preferences from databases of experimentally mapped topologies27 and from endless alignments of homologous sequences.28 That the best predictive methods relied on sequence statistics rather than physicochemical principles as the underlying basis for the prediction has been lucidly highlighted by Bernsel et al.27 These authors proposed a return to basic principles for developing new algorithms27 that take into account an experimental scale of position-specific amino acid contributions to the free energy of membrane insertion.29 Their simplified approach was able to compete in terms of efficiency with the best statistics-based topology predictors. Most importantly, these data demonstrated that the prediction of membrane–protein topology and structure directly from first principles is an attainable goal. Our own contributions to the definition of cholesterol- and sphingolipid-binding domains have the same general objectives: study a particular protein–lipid binding process, understand the basic principles of this interaction, and derive general rules that can be applied predictively to other lipid–protein duets.

1 Introduction

Integral membrane proteins make up a large proportion of the genomes of many organisms—approximately 25% of the human genome—and perform a diverse range of functions, including key steps in the communication of a cell with its environment. Because of their biological and therapeutic importance (Almén, Nordström, Fredriksson, & Schiöth, 2009), membrane proteins are the focus of fundamental and applied biophysical research to characterize three-dimensional structures, dynamics, and interactions in native-like environments.

Solution-state NMR spectroscopy has played a critical role in membrane protein biophysical studies, as the site-specific dynamic and interaction information provided by such approaches nicely complements structural data obtained from X-ray diffraction, cryo-EM, and computational analyses (Cuniasse, Tavares, Orlova, & Zinn-Justin, 2017; Opella & Marassi, 2017). Fundamental to such studies are several 2D “fingerprint spectra,” most often 15N/1H HSQC (heteronuclear single-quantum coherence) spectra (for backbone amide plus Trp, Asn, and Gln sidechains) or methyl 13C/1H HMQC (heteronuclear multiple-quantum coherence) spectra for sidechain methyl groups (Pellecchia et al., 2008). These methyl-directed experiments are especially advantageous for large, slow-tumbling membrane protein/lipid complexes; experiments directed to other sidechain and mainchain sites have been successfully applied as well. NMR experiments can provide information about protein dynamics over many timescales, from fast (ps–ns) sidechain motions to slow conformational changes (μs–ms) (Kasinath, Sharp, & Wand, 2013; Liang & Tamm, 2016; Palmer, 2012; Wand, Moorman, & Harpole, 2013). Many of these dynamics experiments, often using sidechain methyl groups as probes, have been adapted and developed for large biomolecular systems and can be used for membrane proteins (Rosenzweig & Kay, 2014; Sun, Kay, & Tugarinov, 2011; Tugarinov, Hwang, Ollerenshaw, & Kay, 2003).

A particular advantage of solution-state NMR is that proteins are studied in a native-like solution state where they can interconvert among multiple conformations. However, membrane proteins must be solubilized in a suitable membrane mimetic that maintains native structure and dynamics. Different options include detergent micelles, amphipols, bicelles, nanodiscs, SMALPs, and lipid vesicles, each having their own benefits and drawbacks (Liang & Tamm, 2016, 2018; Zhou & Cross, 2013). It is often necessary to test different solubilization strategies for a given protein sample for stability, signal intensity and resolution, and native structure/activity. Two important considerations for all membrane mimetics are (1) a uniform and small particle size and (2) a high extent of deuteration.

High-level deuteration, both within the membrane mimetic and protein itself, is critical to reduce the number of 1H signals present in spectra (including those from lipids, which can be intense) and to improve the relaxation characteristics of the remaining NMR-active spins in the sample. While deuteration is possible for the membrane mimetic through the purchase/synthesis of deuterated compounds, replacing 1H with 2H in proteins requires biosynthetic incorporation. For backbone experiments in eukaryotic expression systems, one can label uniformly with 15N to observe all amides (Eddy et al., 2018; Opitz, Isogai, & Grzesiek, 2015) or through addition of specifically labeled amino acids (Isogai et al., 2016). For methyl groups, one can provide either appropriately labeled amino acids or amino acid precursors (particularly alpha-keto acids) to growth media to access various labeling patterns in the sidechains of several amino acids (Kofuku et al., 2014, 2018). We find isoleucine δ1 methyl groups particularly useful given (1) the abundance of Ile residues in integral membrane proteins including GPCRs (Ulmschneider & Sansom, 2001), (2) the far upfield 13C shift of isoleucine δ1 methyl groups [average 13.5 ± 3.6 ppm 13C according to BioMagResBank (Ulrich et al., 2008)], putting them in a particularly uncrowded region of 2D 13C/1H spectra, (3) the lack of need to stereospecifically assign these methyl groups, unlike Val and Leu, and (4) the presence of multiple, freely rotatable bonds between the methyl group and protein backbone, providing substantial independence of dynamics at these sites (Kasinath et al., 2013).

While many of the aforementioned labeling strategies have been well developed for E. coli, many integral membrane proteins can only be expressed at high levels in eukaryotic hosts. Among these, the methylotrophic yeast Pichia pastoris is a convenient host for heterologous expression and isotopic labeling of eukaryotic membrane proteins (Clark, Dikiy, Rosenbaum, & Gardner, 2018). Advantages of Pichia include rapidity of genetic manipulation, high yields of recombinant protein, existence of posttranslational modification (PTM) and chaperone machinery necessary for eukaryotic membrane proteins, and ability to grow on defined minimal media allowing for perdeuteration (Cereghino & Cregg, 2000; Morgan, Kragt, & Feeney, 2000). Uniform isotopic labeling in Pichia has been well established (Morgan et al., 2000; Pickford & O'Leary, 2004). We have extended this work by demonstrating the 13C, 1H labeling of isoleucine δ1-methyl groups in a perdeuterated background by adding labeled α-ketobutyrate (~ 50% labeling, ~ 90% deuteration) to highly deuterated growth media (Clark et al., 2017, 2015). In contrast, simultaneous labeling of leucine δ- and valine γ-methyl groups with α-ketoisovalerate is inefficient but can be achieved by adding labeled valine directly to the growth media or modifying culture conditions (Clark et al., 2015; Suzuki et al., 2018; Zhang et al., 2017). Pichia can readily take up additional amino acids from media, with a general correlation between uptake efficiency and the energetic cost to synthesize that amino acid type de novo (Heyland, Fu, Blank, & Schmid, 2011). However, after uptake into cells, labeled amino acids can be fed into metabolic pathways (Solà, Maaheimo, Ylonen, Ferrer, & Szyperski, 2004), diluting signal of desired amino acids and complicating data analysis by isotopic scrambling. Alternatively, auxotrophic strains can be developed for labeling a specific amino acid; however, care must be taken to confirm that off-target effects in other metabolic pathways do not arise (Whittaker, 2007).

Here we provide detailed protocols needed to generate such U-2H (13C, 1H-Ile δ1 methyl)-labeled integral membrane proteins by overexpression in Pichia, using the human adenosine A2A receptor [A2AR] as a model system. We further detail how such samples can be used in solution NMR studies, from acquiring simple 13C/1H HMQC spectra, through chemical shift assignments by site-directed mutagenesis, to analyses of 1H–1H cross-relaxation measurements of fast sidechain dynamics.