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Protein-detergent complexes form three-dimensional crystals in which contacts between adjacent protein molecules are made by the polar surfaces of the protein protruding from the detergent micelle257. Although attractive interactions between the micelles might stabilize the crystal packing261,262, these interactions do not lead to rigid crystal contacts. A strategy to increase the probability of getting well-ordered crystals is to attach polar domains to the membrane protein and thereby enlarge the polar surface.

Although the antibody-fragment mediated crystallization of membrane proteins represents a time-consuming and expensive approach, it is a valuable tool for their structural investigation. X-ray structures of several different membrane proteins in complex with antibody fragments have been published263-265. The binding of Fv or Fab fragments increases the hydrophilic part of integral membrane proteins and provides additional surface for crystal contacts. In addition, antigen-antibody complexes can be used to stabilize specific conformations of a protein to aid crystallization attempts. In all reported cases, antibody binding was either essential for the crystallization of the membrane protein or it substantially improved the diffraction quality of the crystals. Up to know all antibody fragments successfully used for co- crystallization were derived from hybridoma cell lines and these antibodies recognize native, nonlinear epitopes of their respective antigen236,266. Two prominent examples are the cytochrome c oxidase263 and the cytochrome bc1 complex264 (depicted in Figure 2.19).

The availability of suitable amounts of highly purified TOM core complex is the basis for three-dimensional crystallization, but only poor-diffracting crystals were obtained. Disorder in the crystals may be caused by the protein existing in different conformations, substoichiometry of its subunits or flexibility. In addition, TOM core complex may exhibit only small hydrophilic domains which are essential for stable crystal contacts. Thus, enlarging the hydrophilic surface and stabilizing defined conformations by binding antibody fragments is a promising approach to obtain well-ordered crystals.

Figure 2.19: Crystal structure of cytochrome bc1 complex. The two monomers of the central

molecule of the crystal are coloured in blue and green, with bound antibody in red264.

Native antibodies are not suitable for co-crystallization attempts. They possess flexible linker regions connecting the variable and constant domains267, and their bivalent binding mode is undesirable. Monovalent antibody fragments can be generated by proteolytic cleavage of the whole antibody, producing two Fab fragments per antibody molecule. These proteolytic fragments are easy to obtain, but care has to be taken to produce homogeneous fragment preparations suitable for crystallization. Residual parts of the flexible linker and, in some cases, glycosylation can hinder crystallization attempts. The crystallization and structure determination of the KcsA K+ channel provides the first example of an integral membrane protein co-crystallized with a proteolytically derived Fab fragment265. Recombinant antibody fragments are more versatile: they can be used in either Fab or Fv format. The different size might be critical for crystallization success, as the bound fragment should outreach the detergent micelle. Antibody-fragments suitable for co-crystallization attempts should bind the protein in its native conformation, have a high binding affinity and form a stable and rigid complex with their native antigen. The last implies the use of antibodies that bind to a discontinuous epitope and therefore, one critical aspect appears to be obtaining antibodies that are native-state specific, i.e., ELISA-positive and Western-negative. In addition, antibodies can help in trapping a flexible protein in a fixed conformation. A variety of structures have been solved in complex with Fv or Fab fragments264,265,268-270. Strikingly, in these cases most or all of the crystal contacts are between the antibodies.

In this study, TOM-directed monoclonal Fv fragments were manufactured by the hybridoma technique and their subsequent bicistronic expression in the periplasma of E. coli237. Although eight different IgG antibodies were obtained, which showed increased affinity in ELISA experiments, the Fv fragment P1C10 showed a much higher affinity upon characterization and was the only fragment which specifically bound to TOM complex. Addition of this antibody fragment to the purified TOM complex in the molar ratio of 5:1 has not been sufficient to saturate the complex, indicating the presence of more than one antibody binding sites. Typically, selection of specific antibodies starts from several thousands of hybridoma clones271. In case of TOM core complex, just a few hundred clones were found, which might be related to the fact that the fusions were frozen for several years before their cultivation took place. The high excess of antibodies from the IgM subclass may result from a biased immunization procedure. Although copurification and cocrystallization of the Fv fragment P1C10 with TOM core complex was successful, no alteration of the crystal quality was observed. Since only one suitable antibody fragment was found, this reduces the probability to achieve an antibody-fragment mediated improvement of the diffraction quality. There is no comprehensive overview of binding constants for all antibodies, which have been co- crystallized with their cognate antigen. Therefore it is not possible to specify yet which affinity threshold is required to successfully obtain well-diffracting crystals of antibody complexes, nor can one rule out the possibility that lower affinities may result in reduced chances of crystallization. In case of the Na+/H+ antiporter NhaA all specific antibodies possess affinities in the nanomolar range, but extensive co-crystallization did not lead to the desired structure determination. This was achieved through the optimization of the purification strategy and other approaches272_.

A promising alternative to raising monoclonal antibodies in mice is the construction of large combinatorial antibody libraries and the adaption of phage display antibody technology for in vitro selection. Another possibility in crystallization of TOM core complex might be to shift to other adaptable binding scaffolds such as the ankyrin repeat using ribosome display for selection141,273,274. For both techniques the selection and production of high-affinity binders are less expensive and time-consuming than for the standard hybridoma technology. The cytochrome c oxidase (COX) from Paracoccus denitrificans was the first successful example to demonstrate the feasibility of the antibody-fragment mediated crystallization approach263. In this case, the co-complex was purified by indirect immunoaffinity chromatography. This strategy could be also pursued for the purification of TOM core complex and might alter its crystallization state.