Two-dimensional crystallization of proteins on lipid monolayers at the air-water interface and transfer to an electron microscopy grid

The two-dimensional (2-D) crystallization of proteins on lipid monolayers a t the air-water interface is a well established method for crystallizing so luble proteins. The transfer of 2-D crystals from the air-water interface t o an electron microscopy (EM) grid constitutes a critical and ill-controlle d step in the whole procedure, which is likely to be responsible for the hi gh variability of results obtained with this method. In this paper, we addr ess the following questions: (1) does the material observed on EM grids con stitute a true representation of the material present at the air-water inte rface? (2) is there an optimal method of transfer to obtain well-ordered pr otein 2-D crystals? To answer these questions, we combine data obtained on three different protein systems, annexin V, streptavidin and cholera toxin, using two types of EM grids, coated with either holey carbon films or cont inuous carbon films. These combined observations help us draw a coherent pi cture of the state of the interfacial films at the air-water surface and pr ovide new insight into the perturbing influence of the transfer step. The m ain conclusions are: (1) both annexin V and streptavidin form crystalline m onolayers at the air-water interface, which are well preserved when transfe r is performed by means of holey carbon films; (2) a major reorganization o f the material present at the water surface accompanies transfer with conti nuous carbon films: the basal monolayer is extensively damaged, transformin g into domains and vesicular structures, which do not pre-exist at the wate r surface; with the three protein systems studied here, these domains are o ften crystalline; (3) the most striking structural reorganization induced b y transfer with continuous carbon films is observed with annexin V, for whi ch the native p6 crystalline assembly is transformed into another crystal f orm, more ordered, with p3 symmetry. It is most probable that these conclus ions also apply to other protein 2-D crystals formed by the lipid monolayer method. The recent in situ observation of 2-D crystals of annexin V formed on solid-supported bilayers, by atomic force microscopy, supports our inte rpretation that monolayers transferred with holey carbon films represent th e genuine material pre-existing at the air-water interface. (C) 1999 Elsevi er Science B.V. All rights reserved.