ABSTRACT
Structural determination of membrane proteins remains a grand challenge in structural biology. Studying the structures of membrane proteins in two-dimensional (2D) membranes that resemble their native environment is a promising alternative to the prevalent method of X-ray crystallography using crystals produced from detergent-extracted membrane proteins in bulk solutions. Here, we explore the possibility of using X-ray scattering to study the structure of membrane-associated proteins in 2D solutions of fluid, single-layered planar lipid membranes that contain these proteins. To illustrate the feasibility of this approach, we review recent results using tobacco mosaic virus adsorbed to a substrate-supported lipid membrane as a model protein. We discuss the information that can be extracted from these data and the prospect of applying these methods to actual membrane proteins. Synchrotron Radiation and Structural Proteomics Edited by Eugenia Pechkova and Christian Riekel Copyright © 2012 Pan Stanford Publishing Pte. Ltd. www.panstanford.com
12.1 INtrodUctIoNX-ray scattering and diffraction are fundamental tools to help structural biologists understand how proteins function by learning about their structures. Synchrotron-based protein crystallography and solution scattering (commonly known as small angle X-ray scattering, or SAXS) have contributed tremendously to structural determination of soluble proteins. These techniques, however, have made little impact in the studies of membrane proteins. This is perhaps not surprising since the native environment of membrane proteins, namely, the cell and organelle membranes, are fundamentally different than that of soluble proteins, i.e., the cytoplasm. Solution scattering measurements are carried out in bulk aqueous solutions that resemble the cytoplasm, and protein crystallography requires samples (crystals) derived from such solutions. Membrane proteins, on the other hand, do not dissolve in water.The prevailing method to make the traditional X-ray scattering and diffraction methods applicable to membrane proteins is to extract the membrane proteins out of the membranes by detergent solubilization and deal with the soluble detergent-protein complex as a whole. Unfortunately, the presence of the detergents greatly hinders the effort to grow high-quality crystals that produce high-resolution X-ray diffraction data. Interpretation of solution scattering data is also never straightforward since the structure of the detergent micelle surrounding the membrane protein is generally not known. More importantly, extracting the membrane proteins out of the membrane necessarily eliminates the possibility to examine how membrane proteins and the membrane interact and how the membrane might regulate the membrane proteins’ function. It is therefore logical to instead study membrane proteins in lipid membranes that resemble their native environment. But doing so will require a set of tools other than traditional protein crystallography and solution scattering.Structural biologists and electron microscopists have already begun to explore structural determination using two-dimensional (2D) membrane protein crystals by reconstituting the membrane proteins back into a lipid bilayer (Hasler et al., 1998; Werten et al., 2002). The obtained 2D crystal is then used in electron diffraction
experiments to determine the structure of the membrane protein. This method is somewhat analogous to X-ray crystallography for soluble protein crystals, in the sense that both permit diffraction-based structural determination using crystals produced in a near-native environment for the proteins, i.e., soluble proteins in aqueous solutions and membrane proteins in lipid bilayers.For soluble proteins, solution scattering is now being used increasingly frequently to deal with proteins that cannot be easily crystallized and to study the structures of proteins in in situ settings. Extending the parallel between soluble protein in bulk solution and membrane proteins in membranes, we will explore using X-ray scattering to characterize the structures of membrane-associated proteins in a solution state as well, with the lipid membrane playing the role of a 2D buffer solution. Like X-ray scattering from bulk solutions, 2D solution scattering will not produce atomic resolution structure. However, it will be able to provide lower resolution molecular envelope and can be used to monitor overall structural changes in membrane proteins in situ, for instance as they bind to ligands. This method can also potentially provide information on the structure of the lipid membrane, as we will discuss below. 2D solution scattering may also be utilized to characterize the interaction between membrane-bound proteins to help optimizing the 2D crystallization process. 12.2 eXIStING StUdIeS2D solution scattering measurements require appropriately prepared membrane samples. The existing studies of membrane structures commonly employ two sample configurations: substratesupported lipid multi-bilayers and Langmuir lipid monolayers. In this section, we review studies relevant to 2D solution scattering and discuss the pros and cons for these sample configurations. We then turn to substrate-supported single layers in the next section, which are rarely used for X-ray studies and but have some important advantages over the other two configurations for the application of structural characterization by X-ray scattering.A well-documented example of X-ray scattering studies of non-crystal structures of membrane-associated proteins is that
on Gramicidin ion-channel and small pore-forming peptides in substrate-supported, multilayered model membranes (see review by Huang and Yang, 2009). The ion-channels or the transmembrane pores induced by pore-forming peptides collectively behave as 2D liquids confined in each of the lipid bilayers within the multilayered stack. X-ray scattering from Gramicidin channels can be observed using lab-based tube X-ray source (He et al., 1993; Yang et al., 1999). Due to the poor scattering contrast, studies of the transmembrane pores usually resorted to neutron scattering with deuterium-hydrogen substitution to enhance scattering contrast. Synchrotron-based experiments have also showed similar scattering patterns from 2D liquids of transmembrane pores (Huang and Yang, 2009). Furthermore, as the sample is dehydrated, the positional correlations between pores located in neighboring bilayers start to develop and the pores can form 3D periodic structures that diffract like a crystal. The structures of the pores formed by alamethicin (Qian et al., 2008a) and a Bax-α5 (Qian et al., 2008b) in Br-containing lipid bilayers have recently been solved using the multi-wavelength anomalous dispersion method at Br K-edge.A major drawback of structural studies utilizing lipid multi-bilayers is that the samples are measured in a partially dehydrated state in a humidified environment, where the water vapor surrounding the sample maintain a water layer that is ~1 nm thick between bilayers. The chemical condition in the inter-bilayer water is not well defined and cannot be easily characterized or controlled. This method therefore is not suitable for generic membrane-associated proteins, which may require specific chemical environment to function. Small peptides can be introduced into the supported lipid multilayer by pre-mixing the organic solutions of lipids and peptides when preparing the multilayer. This again may not be feasible for membrane-associated proteins in general. For easier introduction of proteins and control of the chemical environment around the proteins, Langmuir monolayers are often utilized. Langmuir monolayers are single-leaflet lipid membranes spread at the air-water interface. Proteins can be introduced into the aqueous subphase and interact with the lipid membrane. Most X-ray scattering studies of proteins adsorbed to a Langmuir monolayer focused on specular reflectivity (Vaknin et al., 1993;
Haas and Moehwald, 1994; Weygand et al., 1999; Zheng et al., 2001), which provides information only on the average structure along the direction normal to the monolayer. Clearly, more information can be extracted from off-specular scattering due to the in-plane structure. For instance, in a recent study of poorly ordered crystals of cholera toxin (Miller et al., 2008), the qz-dependence of the diffraction intensity along the off-specular quasi-Bragg rods (qz is the component of the scattering vector normal to the membrane) was analyzed to infer the molecular orientation of the bound proteins with respect to the membrane.Under appropriate condition in the subphase, the proteins adsorbed to the Langmuir monolayer can form 2D crystals (Kornberg and Darst, 1991; Dietrich and Vénien-Bryan, 2005). Grazing incidence X-ray diffraction from such 2D protein crystals has been demonstrated (Haas et al., 1995; Lenne et al., 2000). While structural reconstruction has not been reported using diffraction data obtained from these 2D crystals, Lenne et al. were able to collect from 2D streptavidin crystals diffraction data corresponding to 10 Å in-plane resolution (Lenne et al., 2000). It should be noted that 2D protein crystals are usually very small (for instance, lateral dimension of streptavidin 2D crystals can be as large as tens of microns). Therefore, in these grazing incidence diffraction measurements X-ray diffraction comes from an ensemble of crystals with random in-plane orientations within the illuminated area, which is usually centimeters long along the beam direction. This could pose a problem for structural determination since Bragg rods at identical or similar qr positions may not be resolved (qr is magnitude of the component of the scattering vector parallel to the membrane). 12.3 SUbStrate-SUpported, SINGle-layered
lIpId membraNeSSubstrate-supported single-layered lipid membranes submerged under a buffer solution present a unique opportunity for X-ray scattering studies of the structure of membrane-associated proteins. These membranes can be prepared in different configurations to accommodate various types of membrane-associated proteins
(Fig. 12.1a-c). Like Langmuir monolayers, these lipid membranes are in direct contact with the bulk aqueous solution, which provides a means to control the chemical environment and incorporate proteins into the membranes. FRAP measurements have shown that the lipid molecules in these supported membranes remain mobile (Boxer, 2000; Yang et al., 2009), as in native biological membranes. The mobility of lipids, which is essential for the biological function of the proteins, is also required to create 2D crystals of lipid-bound proteins (Kornberg and Darst, 1991; Mosser and Brisson, 1991; Weisenhorn et al., 1992; Dietrich and Vénien-Bryan, 2005). For instance, 2D streptavidin crystals formed on supported lipid bilayers
Figure 12.1 Schematic representation of X-ray scattering from 2D solution of membrane-associated proteins. The proteins are embedded in a substrate-supported lipid membrane, which in turn is submerged in a buffer solution that provides a suitable chemical environment for the protein to function. The lipid membrane and the proteins can be associated in several different configurations. (a) A substrate-supported bilayer is suitable for transmembrane proteins that do not extend beyond the thickness of the lipid bilayer. (b) Peripheral proteins that only penetrate into one leaflet of the bilayer can be studied in a lipid monolayer supported by a hydrophobic substrate (e.g., Plant, 1999; Scheuring et al., 1999). (c) In the most general case, a complete bilayer is assembled above the substrate-supported monolayer. Transmembrane proteins that are embedded in the lipid bilayer are constrained to the monolayer by Ni-His tag linkers. This process has been used with Langmuir lipid monolayers to create 2D membrane protein crystals (e.g., Hasler et al., 1998; Lévy et al., 1999). His tags are commonly present in recombinant membrane proteins due to the requirement for the purification process and Ni-chelating lipids are commercially available. See also Color Insert.
