In Cubo Crystallization

In contrast to the original human p2AR [2] and turkey p1AR [4] , which were crystallized using hanging drop vapor diffusion, both the p2AR and A2a T4L

Bicontinous
Figure 14.4 Comparison of the structures of the PAR showing the positions of the antibody and T4L insertion used to facilitate crystallization.

fusions were crystallized using the cubic phase method to grow crystals. To date, 54 structures have been determined from crystals grown using the cubic phase method, which represents approximately 10% of the total membrane protein structures deposited in the PDB, although the majority of these are homologues and mutants of bacteriorhodopsin [177]. Moreover, the use of the cubic phase method has resulted in some of the highest resolution GPCR structures to date (e.g., halobacterial bacteriorhodopsin was solved to 1.43 A) [178]. Cubic phase has also been shown to be compatible with detergent-free membrane protein crystallization in which bacteriorhodopsin crystals have been directly grown from native cell membranes without exposure to any detergent [179]. While cubic phase is a relatively recent development in protein crystallography, it offers a real opportunity to further develop the field of GPCR structural determination. The basic premise behind in cubo crystallization is that membrane proteins crystallize in a more native-like lipid bilayer, as opposed to a detergent micelle. This relies on the protein being incorporated into an appropriate lipidic matrix [180] , without altering its native structure, allowing diffusion in three dimensions to allow for nucleation and crystal growth (Fig. 14.5) [177,181].

The lipidic matrix of choice is a bicontinuous cubic phase of monoacylglyc-erols (MAGs) and water. MAGs are an important intermediate in fat metabolism that adopts a remarkable variety of liquid crystal phases when dispersed in water; this makes them an ideal matrix choice. In both the recent p2AR and A2a T4L fusion structures, the MAG used was monoolein, which was substantially supplemented with cholesterol. While cholesterol provides an active component required to stabilize proteins, the lipid also provides a large number

Figure 14.5 Schematic representation of the events proposed to take place during the crystallization of a GPCR from the lipidic cubic mesophase. Crystallization is initiated with the protein being reconstituted into the highly curved bilayers (bottom left-hand quadrant of the figure). The addition of "precipitants" then shift the equilibrium away from stability in the cubic membrane, leading to phase separation wherein protein molecules diffuse from the continuous bilayered reservoir of the cubic phase by way of the lamellar portal (left upper quadrant of figure) to lock into the lattice of the advancing crystal face (right upper quadrant of figure). Salt (positive and negative signs) facilitates crystallization by charge screening. Co-crystallization of the protein with native lipid (cholesterol) is shown in this illustration. As much as possible, the dimensions of the lipid, detergent, native membrane lipid, protein (p2AR-T4L; PDB code 2RH1), bilayer, and aqueous channels have been drawn to scale. The lipid bilayer is approximately 40-A thick. The figure was kindly provided by Professor Martin Caffrey, University of Limerick [207].

Figure 14.5 Schematic representation of the events proposed to take place during the crystallization of a GPCR from the lipidic cubic mesophase. Crystallization is initiated with the protein being reconstituted into the highly curved bilayers (bottom left-hand quadrant of the figure). The addition of "precipitants" then shift the equilibrium away from stability in the cubic membrane, leading to phase separation wherein protein molecules diffuse from the continuous bilayered reservoir of the cubic phase by way of the lamellar portal (left upper quadrant of figure) to lock into the lattice of the advancing crystal face (right upper quadrant of figure). Salt (positive and negative signs) facilitates crystallization by charge screening. Co-crystallization of the protein with native lipid (cholesterol) is shown in this illustration. As much as possible, the dimensions of the lipid, detergent, native membrane lipid, protein (p2AR-T4L; PDB code 2RH1), bilayer, and aqueous channels have been drawn to scale. The lipid bilayer is approximately 40-A thick. The figure was kindly provided by Professor Martin Caffrey, University of Limerick [207].

of the crystal-packing interactions between symmetry-related protein molecules, as observed for the p2AR. The interfaces between the p2AR molecules contained six cholesterol and two covalently bound palmitic acid molecules, with the latter further illustrating the requirement for a homogeneous protein. This further highlights the role in which PTMs can play in facilitating crystallization [2]. Although monoolein has typically been the most favored MAG

used in cubic phase, the use of more rationally designed MAG matrices, all of which allow the formation of the requisite cubic mesophase at room temperature required for crystallization, has been sought [177].

The unique structure of the cubic phase means that it differs in a number of ways from that of the more classic solution phase (hanging drop vapor diffusion crystallization). Upon reconstitution into the bilayer of the cubic phase, integral membrane proteins such as GPCRs become confined to the 3D network of the curved bilayers, through which they diffuse freely (Fig. 14.5). Concurrently, small molecules, such as precipitants and buffers, diffuse through the adjacent network of aqueous channels [182, 183]. Diffusion conditions may be further complicated around the crystal, where the lipid phase forms a stack of lamellar layers [183]. In contrast, during solution-phase crystallization, both the protein molecules and the small molecule precipitants freely diffuse through the same 3D space.

It is thought that in cubo crystallization is mediated through the development of a series of overlapping concentration gradients within the porous mesophase. Upon reconstitution, the protein diffuses within the plane of the cubic phase bilayer, and, as precipitant is added to the mesophase, this triggers phase separation. Under conditions favoring crystallization, one of the separated phases is enriched in protein, facilitating protein-protein and protein-Hpid contacts, which nucleate and develop into crystals . 177] . The m cubo model also includes a lamellar conduit between the bulk cubic phase acting as a protein reservoir and the face of the crystal [184].

For the future development of GPCR crystallization, the use of new and emerging technologies will be required in order to maintain this momentum. The development of new techniques to set up crystallization trays with minimal amounts of protein, such as microfluidic protein crystallization systems [185], will dramatically increase the range of conditions that can be screened with relatively small quantities of protein. Furthermore, the application of in cubo crystallization will likely allow further structural determination of GPCRs. Finally, the development of microdiffraction technology [186], which has been a crucial component in all of the recent GPCR structures [2, 4, 5] . will hopefully become more widely available in the future.

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