Large single crystals of the Neurospora crassa plasma membrane H+-ATPase: an approach to the crystallization of integral membrane proteins.

Large single crystals of the dodecylmaltoside (DDM) complex of a polytopic integral membrane transport protein, the Neurospora plasma membrane H(+)-ATPase, have been obtained using an approach that attempts to take into account the possibly radically different physicochemical properties of the protein surfaces and the detergent micellar collar. The overall goal of the crystallization strategy employed was to identify conditions in which the protein surfaces of the DDM-ATPase complex are moderately insoluble and in which the DDM micellar collar is also near its solubility limit. The first step was to screen a variety of commonly used protein precipitants for those that were able to induce the aggregation of pure DDM micelles. The concentration at which any precipitant induced DDM micellar aggregation was hoped to be close to the concentration at which it might induce insolubility of the detergent micellar collar of the DDM-ATPase complex. Of the nine precipitants tried, seven, all polyethylene glycols (PEGs), were able to induce DDM micelle insolubility. The seven PEGs were then tested for their effect on the solubility of the DDM-ATPase complex at a concentration slightly below that necessary to induce DDM micellar aggregation. Three of the PEGs caused extensive precipitation of the ATPase at this concentration and were, therefore, shelved. The other four PEGs did not induce precipitation at the concentration employed and were subsequently used at this concentration for crystallization trials in which the protein concentration was varied. Encouragingly, crystalline plates of the ATPase were obtained for each of the four PEGs tried, indicating that the overall approach may be valid. Unfortunately, the crystals obtained were visibly flawed, suggesting that the correct balance of protein surface and DDM micelle insolubility had not yet been reached. The ionic strength of the crystallization trials was then raised, which was known from other experiments to render the protein surfaces of the ATPase less soluble while having no effect on the DDM micellar aggregation point. For one of the PEGs, PEG 4000, this brought on a new, well formed hexagonal crystal habit. Subsequent optimization of the initial conditions has yielded large single hexagonal crystals of the H(+)-ATPase roughly 0.4 x 0.4 x 0.15 mm in size, holding promise for exploration of the structure of the ATPase by X-ray diffraction analysis.