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Membrane Protein Structures by X-Ray Crystallography Membrane Protein Nuclear Magnetic Resonance Spectroscopy Membrane Protein Structures by Electron Microscopy Imaging Membrane Proteins by AFM Summary
The biological importance of membrane proteins has been recognized worldwide for many years, but historically these proteins have proved difcult to characterize structurally because of a variety of experimental challenges. Recently, technological advances across several disciplines have prompted considerable progress in three-dimensional structure determination of membrane proteins. This review describes the state-of-the-art methods that have successfully produced high-resolution membrane protein structures to date. Most notably, X-ray crystallography will be discussed, as this technique has made by far the largest contribution to our current knowledge of membrane protein structure. This is followed by discussion of nuclear magnetic resonance spectroscopy and cryo-electron microscopy, both techniques that have also been successful in producing high-resolution structures for membrane proteins, albeit to a lesser degree than X-ray crystallography. Finally, we will discuss atomic force microscopy. Although this technique cannot be used for atomic level structural determination, it offers distinct advantages for investigation of membrane protein oligomerization, dynamics, and large-scale conformational changes. Recent notable membrane protein structures are included throughout to illustrate progress in the eld as well as the strengths and weaknesses of each method.
Structural, biophysical, and biochemical studies of membrane proteins have revealed the importance of this class of proteins in fundamental biological processes such as the import and export of nutrients and waste into and out of cells, cell division, and signaling, to name a few. With approximately one third of sequenced genomes encoding integral membrane and membrane-associated proteins, and two thirds of all drugs in development targeting membrane proteins, their importance is now well accepted. However, three-dimensional (3-D) structures of the majority of known membrane proteins still elude us. Of the 47,000 protein structures deposited in the Protein Data Bank (PDB), only about 1% are for membrane proteins. Solving the structures of these proteins has proved to be highly challenging, and it still represents the leading edge of protein structural biology. The slow progress is caused, in part, by difculties in protein production and purication (especially for eukaryotic membrane proteins) and in part by experimental difculties encountered because of their large
size and the requirement for detergent/lipid solubilization. Despite these obstacles, signicant advancement has been made over the past decade. Careful production of membrane proteins and major breakthroughs in instrumentation have yielded stunning results for several classes of membrane proteins, including channels, pores, and receptors. The resulting structural data have given us an insight into the architecture of both simple and complex membrane proteins as well as their function (for an excellent summary of known structures, see http://blanco.biomol.uci.edu/Membrane Proteins xtal.html ). In this review, we will cover the four most successful and commonly used experimental methods for determination of membrane protein structures, namely X-ray crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and atomic force microscopy, with particular emphasis on recent advances. We note that molecular dynamics simulations have also contributed signicantly to our current understanding of membrane protein structure, and closely accompany the above 1
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listed methods; however a description of these computational methods is beyond the scope of this review.
and the recently developed lipid-phase methods. Detailed information on membrane protein crystallization can be found in several excellent reviews in the Further Reading section. The general principle of protein crystallization involves the supersaturation of a protein solution. The addition of precipitating agents such as salts, organic solvents, or polymers triggers the crystallization process. Detergents play a vital role in the crystallization of membrane proteins. However, as mentioned, they can also have a destabilizing effect on the protein. For successful crystallization, the detergent micelles must be accommodated in the crystal lattice with minimum effect on the formation of crystal contacts. The choice of detergent and its associated properties (polarity of head group, aliphatic chain length, size) must be explored for each protein (3). Generally, charged detergents should be avoided because of the risk of repulsion between protein-detergent complexes. The length of aliphatic chains should be balanced between the need to cover the proteins hydrophobic surfaces and the need to have as low a detergent volume as possible to maximize protein-protein contacts. The phase behavior of detergents and lipids is also a pertinent consideration. Phase separation from a micellar phase (detergent-rich) toward a nonmiscible phase (detergent-poor) can occur at high-detergent and precipitant concentrations. These phase boundaries have been exploited to enhance crystallization of membrane proteins (4). The use of additives such as small amphiphiles (e.g., heptane-triol, LDAO) can also enhance crystallization by reducing the volume of detergent in the protein-detergent complex, leading to increased numbers of crystal contacts (5). However, the overriding problem with the use of detergents is the increase in the number of variables that need to be optimized in crystallization trials, which is already considerable. Coupled with the use of additives, this presents a huge number of possibilities. The absence of a general set of rules to guide detergent/additive choice is currently a signicant problem for crystallization, and it increases the timescale for each structure. A signicant landmark in membrane protein crystallization has been the development of lipidic cubic phase technology (6). This technically challenging approach is capable of pro while providing an ducing crystals that diffract beyond 2 A environment resembling that of a natural membrane. Lipidic cubic phases are gel-like materials into which the protein is embedded. Crystallization is then initiated by the addition of precipitants. If successful, the resulting crystals contain ordered layers of protein-lipid sheets, with contacts formed within and between the sheets. Lipidic cubic phases have been successfully used to crystallize several membrane proteins, including archeal seven transmembrane proteins (6, 7). Co-crystallization with antibody fragments (Fv fragments of Fab domains) has also been used to enhance membrane protein crystallization (8, 9). The fragments form bridges between protein-detergent complexes, thus increasing the number of protein-protein contacts. Using this method, crystallization was achieved for the cytochrome bc1 complex (8), the KcsA potassium channel (10), and more recently the human -2 adrenergic receptor (Fig. 1a) (1113). A caveat to this approach is that antibody production must be
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tailored to the protein under study, which can be expensive and time consuming. The process of crystallization is still largely a process of trial and error, but in an attempt to formulate general rules, the cumulative experiences of X-ray crystallographers are being compiled into online databases such as the Marseille Protein Crystallization Database (14) and the Biological Macromolecule Crystallization Database (15). These and similar databases will provide useful starting points in crystallization trials and may greatly accelerate the process.
Sodium-potassium pump
The structure of the sodium-potassium pump was solved in (Fig. 1b), in complex with two 2007 to a resolution of 3.5 A bound rubidium ions (13). Structures were also solved for other members of the P-type ATPase family (18, 19) around the same time revealing the structural similarity between family members as well as the conformational changes that occur during function.
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
(a)
(b)
(c)
Figure 1 Ribbon representations of three membrane protein structures determined using X-ray crystallography. (a) The human -2 adrenergic receptor was crystallized using the technique of antibody cocrystallization (11) (PDB ID: 2R4R). The structures of the receptor (i) and the antibodies (ii) are shown (b) The structure of the sodium-potassium pump (12) (PDB ID: 3B8E) is shown, indicating the three subunits of the protein. (c) The structure of the acid-sensing ion channel 1 at low pH (13) (PDB ID: 2QTS), which is composed of three chains (i, ii, and iii) that assemble to form a trimer.
Solid-state NMR
Solid-state NMR will only be considered in brief because of the limited number of novel membrane protein structures produced. For comprehensive reviews of this technique, the reader is referred to the Further Reading section. Around 17 structures (mainly small peptides) solved by solid-state NMR have been deposited in the PDB, with the rst being gramicidin A in 1997 (21) and the most recent being the backbone structure of Inuenza A M2 proton channel transmembrane domain (22). Solid-state NMR differs from the solution-state in that the molecule under investigation is static or slowly tumbling, resulting in broad NMR peaks with low resolution and sensitivity. This fundamental problem has been tackled by development of MAS (magic-angle spinning) (23) and REDOR (rotational echo double resonance) (24) experiments. Advantageously, solid-state NMR methods enable membrane proteins to be studied in lipid bilayers resembling native membranes; a disadvantage is that proton detection is challenging, with most studies focusing on isotopically labeled samples.
produce high-resolution spectra, and maintain the protein in its native conformation. Detailed protocols for protein and sample preparation can be found in the Further Reading section. Detergents are frequently used to solubilize membrane proteins for solution-state NMR. Rapidly tumbling protein-micelle complexes are formed (Fig. 2a) (2527) that are capable of producing narrow linewidths and high-resolution spectra while maintaining the quaternary structure of the protein. A detergent screen is often required to nd the optimum detergent for a particular protein, and extensive studies of detergents suitable for NMR have been performed (28). Although no detergent is generally applicable, in numerous cases, dodecylphosphocholine (DPC) and lyso-palmitoyl phospatidylglycerol (LPPG) have produced high-quality solution spectra, providing a strong starting point in detergent screens (28). The use of detergents is currently limited to proteins of <40 kDa (protein-micelle complexes of <80 kDa). Alternatives to detergents have also been employed with moderate success. Organic solvent mixtures have proved useful (29) and offer the added advantage that proteins tumble more rapidly producing higher quality spectra. However, these mixtures are considered poor membrane mimetics, casting doubt on the relevance of resulting structures. Bicelles (Fig. 2b), or lipidic disk-shaped particles that more closely resemble bilayers, have also been useful in studies of small transmembrane peptides and can be used in both solution and solid-state NMR (30). Further solubilizing agents include lipopeptides and amphipols (Fig. 2d), which have been used in initial studies of OmpA, PagP, and DAGK (3133), whereas nanodisks (Fig. 2e) (34) and lipid cubic phases (Fig. 2f) (35) are recent developments that may prove useful in the future.
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(a)
(b)
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(d)
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Figure 2 Membrane mimetics used in NMR studies of membrane proteins. (a) Small, spherical detergent micelles composed of amphiphillic detergent monomers. Dodecylphosphocholine (DPC) micelles were employed to determine the structure of OmpA (25) (PDB ID: 1G90) (b) Bicelles, composed of lipid/detergent mixtures, have been used recently to solve the structure of the Bnip3 transmembrane domain dimer (26) (PDB ID: 2J5D) (c) Synthetic lipid bilayers closely approximate the natural environment of membrane proteins and have been used with solid-state NMR to determine the structure of the fd bacteriophage pVIII coat protein (27) (PDB ID: 1MZT). (d) Amphipols are amphiphillic polymers consisting of a hydrophilic backbone onto which numerous hydrophobic chains are attached that can coat the hydrophobic region of membrane proteins. (e) Nanodisks are self-assembling structures composed of a phospholipid bilayer disk encircled by an engineered membrane scaffold protein. (f) Lipid cubic phases are primarily used in X-ray crystallography studies but recently have shown promise for use in NMR studies as well. (g) NMR structure of the phospholamban pentamer (PDB ID: 1ZLL), looking down on the central cavity. (h) Side view of the phospholamban pentamer shown in (g) overlaid with a rened version of the structure published in 2006 (PDB ID: 2HYN).
from the development of the TROSY (transverse relaxation optimized spectroscopy) triple resonance experiments, which improve spectral resolution and sensitivity (36). Although TROSY experiments will not be covered in detail here, briey these experiments reduce relaxation of NMR signals at high magnetic eld strengths, thus increasing the molecular mass of proteins that can be studied to 50 kDa. Additional benets can be realized by using TROSY in conjunction with a selectively deuterated protein and deuterium decoupling, acting to further reduce broadening of signals (37). TROSY experiments have been used successfully to assign the resonances of both -barrel (25, 38) and -helical membrane proteins (39). After completion of the spectral assignment, NMR-derived short- and long-range distances can be determined by measurement of nuclear Overhauser enhancements (or NOEs). An NOE apart, and the is an interaction between a pair of atoms 5.0 A
intensity of the NOE can be related to the distance (r) separating the pair. The use of NOEs can be problematic for helical proteins as they tend to have few long-range NOEs. The use of methyl protonation can increase the number of NOEs; however, because of the poor chemical shift dispersion of methyl groups in helical membrane proteins, this approach is generally ineffective. An alternative is the use of residual dipolar couplings (RDCs) to obtain long-range distances. RDCs are derived from the difference in coupling constants in an aligned and unaligned state, providing information on the orientation of internuclear vectors relative to the external magnetic eld (40). Several methods have been developed to produce weak alignment of membrane proteins, thus enabling the measurement of RDCs. Polyacrylamide gels can be used for small-to-medium proteins to produce an anisotropic environment where protein orientations are limited, resulting in weak alignment (40). 5
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DNA nanotubes are also a promising alignment method with the added advantage that they are resistant to detergent and do not reduce achievable protein concentrations (41). Alignment can also be achieved by incorporation of paramagnetic lanthanide ions into diamagnetic proteins (42). Once aligned samples are prepared, RDCs are accurately measured using a set of TROSY-based experiments (43) Importantly for helical proteins, a plot of backbone amide RDCs versus residue number produces a wave pattern with a periodicity of 3.6 residues per cycle for helical regions, allowing detection of helical secondary structure and the tilt of the helix relative to the magnetic eld (44). The use of RDCs for structure renement has been demonstrated for several membrane proteins, including OmpA (45), Vpu (46), and MerF (47). Finally, NMR-derived distance information as well as information about dihedral angles (obtained from chemical shifts) is incorporated into structure calculations performed using molecular dynamics and simulated annealing programs such as CNS (48) and XPLOR-NIH (49) to calculate the protein structure.
Phospholamban pentamer
Phospholamban is a homopentameric membrane protein involved in muscle contraction through regulation of the calcium pump in cardiac muscle cells. The structure of the unphosphorylated protein solved in DPC micelles reveals a symmetric pentamer of phospholamban monomers (Fig. 2g) stabilized by leucine/isoleucine zipper motifs along the transmembrane domains (51). Notably, another structure was produced for phospholamban (Fig. 2h) that used a variant of the traditional simulated annealing and molecular dynamics protocol that reduced the chances of entrapment in local minima (52). 6
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of choice for structure determination by EM, and it is used almost exclusively in recent reports. Additional improvements in the resolution of cryo-EM data are achieved by imaging samples present in 2-D crystals, a subject that will be covered in more detail in the next section.
suitable lipids and lipid:protein ratios is required (67) to obtain a high-quality crystal. However, in some cases, membrane proteins that have proved very resistant to 3-D crystal formation have readily formed 2-D crystals in lipid membranes. Two types of 2-D crystal have been analyzed by cryo-EM: sheet-like (planar) crystals and tubular crystals. Planar crystals are tilted in the electron beam in order to obtain different projection maps of the crystal at a variety of angles. Maps collected at different angles are then averaged together to produce a 3-D reconstruction (68). However, the planar crystal cannot be tilted through all angles in the microscope (for example, a sample rotated through 90 would then be parallel to the electron beam) and typically can only be rotated through 7075 . The resulting resolution is, therefore, spatially heterogeneous and appears to have what is known as a missing cone of data. Alternatively, membrane proteins can also crystallize into tubular crystals where, in a single crystal tube one can potentially see all orientations of a protein without the need to tilt the crystal. Tubular crystals overcome the missing cone of information and provide resolution that is equal in all directions (59).
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conformational changes of a protein over time, thus yielding critical insight into function. In these time-resolved studies, conformational changes are induced in the protein (by changing pH, adding ligand, etc.) and then trapped by freezing the crystal. Freezing can take place at several time points after induction of a conformational change to produce a series of structures that describe a reaction pathway. Time-resolved studies have been performed to investigate the photocycle of bacteriorhodopsin (72), the gate-opening mechanism of the acetylcholine receptor (57), and more recently, the pH-dependent mechanism for a Na+ /H+ antiporter (73).
Aquaporin-4
structure of Aquaporin-4 (Fig. 3c) was determined This 3.6-A by electron crystallography of double-layered 2-D crystals (56). Features in the structure show that Aquaporin-4 can form membrane junctions, and they suggest for the rst time its role in cell adhesion. This structure is of additional interest in that it is the rst structure of a multispanning mammalian membrane protein obtained by purely recombinant methods.
Glutathione transferase-1
the resolution of this recent structure also rivals that At 3.2 A, obtained in XRC (63). This structure provides a strong insight into the function of the protein, and it reveals key differences between the glutathione binding site of this membrane-spanning enzyme and that of its soluble counterparts.
(a)
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Figure 3 Ribbon representations of three membrane protein structures determined using electron microscopy. (a) The structure of Aquaporin 0 AQP0 (53) (PDB ID: 2B6O), the highest resolution structure of a membrane protein to be solved by cryo-EM to date. (b) A view down the pore of AQP0 (arrow), which allows water molecules to pass across the membrane. (c) The rat AQP4 (56) (PDB ID: 2D57) structure, highlighting the gap (arrow and *) between two helices that align to span the membrane.
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(a)
(b)
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(d)
Figure 4 High-resolution AFM images of the surface of the KirBac3.1 potassium channel in a 2-D crystal in the presence of EDTA (a) or Mg2+ (b) allow us to watch the channel open and close. The channel is in its open state in the absence of Mg2+ , and that can be seen clearly from the image shown in (c), across. (d) This cavity is no longer visible in the image of KirBac3.1 in its closed state, which is a result of adding which reveals a central cavity 30 A Mg2+ . (Reprinted from Reference 76 with permission from Elsevier.)
in order to produce a contour map of its surface. In order to maximize resolution, vertical uctuations of the tip must be minimized. This is achieved using a self-regulating feedback system (or servo system), which keeps the cantilever deection approximately constant by making small adjustments in the vertical displacement of the sample. A 2-D crystal provides an ideal sample for AFM as it is hard and at, with the protein embedded in a densely packed array, thus restricting any lateral or vertical movement of the protein. Another prerequisite for high-resolution data is minimal interaction between the tip and the sample, and this is greatly inuenced by the size and the geometry (or sharpness) of the tip. The tip geometry is most commonly an inverted pyramid. Depending on its sharpness, as the tip moves across a surface, structures may interact with both the end of the tip as well as the sides of the tip. Any interactions with the sides of the tip will cause a broadening of the signal, thus reducing the lateral resolution of the image. This effect, also known as tip convolution, is most noticeable on surfaces with considerable height differences. Most of the tips used on high-resolution instruments are now commercially fabricated, with the best tips having a radius of curvature of >5 nm.
protein is attached by contact adhesion, the tip is then retracted. Force versus distance curves are recorded before and after adhesion in order to determine the forces involved in unfolding or unzipping the protein. In addition to the force versus distance curves, images can also be acquired before and after an unfolding event to observe the effects on the surrounding environment (85).
F0 F1 -ATP synthase
The F0 F1 -ATP synthase is responsible for synthesizing ATP in many organisms. This enzyme is composed of two rotary motors (F0 and F1 ) connected by a central stem, and the stoichiometry of these two motors is of critical importance to energy conversion. F0 F1 -ATP synthases are very large, making them unsuitable for study by NMR or X-ray crystallography. However, AFM is ideally suited to such applications and has been used to determine the oligomeric states of the F0 and F1 motors in several species of bacteria and plants to shed new light on how the enzymes function (84, 86, 87).
Summary
The aim of this review has been to summarize the key techniques currently used for determination of membrane protein structures. Thus far, XRC has produced the largest number of 9
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membrane protein structures, and it has achieved the highest resolution of any other technique. However, this technique still requires a high-quality crystal, which is currently the rate-limiting step in the process. Steady progress in our understanding of crystal growth and the development of new crystallization methods show great potential for future studies. NMR has proved to be a valuable technique for the study of small membrane proteins and individual domains of larger proteins. In addition to 3-D structural information, NMR can also provide information on dynamics and ligand binding. NMR also has the advantage that it does not require crystals, which are difcult and time-consuming to produce, and instead uses a large range of membrane mimetics and solubilizing agents. The major limitations of NMR remain the upper limit on protein size and difculties in resolving resonances for assignment purposes. Continued developments in instrumentation, experimental methods, and improved membrane mimetics will greatly advance the eld. EM has also made a large impact on our knowledge of membrane proteins and promises to equal or surpass the more static structural methods of XRC and NMR. Because of recent new developments in technology, cryo-EM has caught up with XRC and NMR in terms of resolution and is well positioned to lead the eld in the future. Both EM and AFM offer the unique advantage that membrane proteins can be studied in synthetic lipid bilayers as well as their native membranes, producing more biologically relevant structural data. Furthermore, these methods allow study of conformational changes over time, in response to such events as ligand binding or unfolding. However, the most exciting prospect for future studies of membrane proteins comes when data from all these methods are used in concert, alongside molecular dynamics simulations, to describe the structures and functions of these essential proteins.
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Further Reading
Caffrey M. Membrane protein crystallization. J. Struct. Biol. 2003;142: 108132. De Angelis AA, Jones DH, Grant CV, Park SH, Mesleh MF, Opella SJ. NMR experiments on aligned samples of membrane proteins. Meth. Enzymol. 2005;394:350382. Engel A. Robert Feulgen Lecture. Microscopic assessment of membrane protein structure and function. Histochem. Cell Biol. 2003;120: 93102. Fernandez C, Wider G. TROSY in NMR studies of the structure and function of large biological macromolecules. Current opinion in structural biology. 2003;13:570580. Janovjak H, Kedrov A, Cisneros DA, Sapra KT, Struckmeier J, Muller DJ Imaging and detecting molecular interactions of single transmembrane proteins. Neurobiol. Aging 2006;27:546561. McPherson A. Introduction to protein crystallization. Methods 2004;34: 254265. Mosser G. Two-dimensional crystallogenesis of transmembrane proteins. Micron. 2001;32:517540. M uller DJ, Sapra KT, Scheuring S, Kedrov A, Frederix PL, Fotiadis D, Engel, A. Single-molecule studies of membrane proteins. Curr. Opin. Struct. Biol. 2006;16:489495. Prive GG. Detergents for the stabilization and crystallization of membrane proteins. Methods 2007;41:388397. Qian B, Raman S, Das R, Bradley P, McCoy AJ, Read RJ, Baker D. High-resolution structure prediction and the crystallographic phase problem. Nature 2007;450:259264. Sanders CR, Sonnichsen F. Solution NMR of membrane proteins: practice and challenges. Magn. Reson. Chem. 2006;44:S2440. Smyth MS, Martin JHJ. X-ray crystallography. J. Clin. Pathol.-Mol. Pathol. 2000;53:814. Tamm LK, Liang B. NMR of membrane proteins in solution. Prog. Nucl. Magn. Reson. Spectr. 2006; 201210. Watts A, Straus SK, Grage SL, Kamihira M, Lam YH, Zhao X. Membrane protein structure determination using solid-state NMR. Meth. Mol. Biol. 2004;278:403473. Werten PJ, Remigy HW, de Groot BL, Fotiadis D, Philippsen A, Stahlberg H, Grubmuller H, Engel A. Progress in the analysis of membrane protein structure and function. FEBS Letts. 2002;529: 6572. Wiener MC. A pedestrian guide to membrane protein crystallization. Methods 2004;34:364372.
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WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
See Also
Protein Expression, Systems for Solubilize Membrane Proteins, Techniques to Crystallization of Proteins, Overview of Applications in Chemical Biology NMR for Proteins Imaging Techniques for Proteins
WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.
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