FORMATION OF 2 DIMENSIONAL PROTEIN CRYSTALS FOLLOWED BY ATOMIC FORCE MICROSCOPY

Ilya Reviakine, Wilma Bergsma-Schutter, Alain Brisson

I.Reviakine@chem.rug.nl

Electron Microscopy group Biophysical Chemistry, University of Groningen, Nijenborgh, 4, 9742 AG Groningen, the Netherlands

Keywords: Crystal growth, Protein 2-D crystallisation, Atomic Force Microscopy, Supported planar lipid bilayers, Annexin V.

In electron crystallography 2 dimensional (2D) crystals are used for determining the structure of (macro)molecules. A well established method for 2D crystallisation of soluble proteins is the so-called lipid monolayer technique, where 2D crystals are formed on a lipid monolayer incorporating a protein-specific ligand spread at the air-water interface (1,2). While the technique has been used successfully to crystallise a number of proteins, the understanding of the crystallisation process itself was hindered by the lack of experimental approaches suitable for its investigation. Advent of several new techniques - like Atomic Force and Brewster angle microscopies - has already lead to new developments and promises a greater insight into this area. We have utilised the unique ability of AFM to provide molecular-resolution images of biological macromolecules in their native aqueous environment to follow the formation of 2D protein crystals in situ and in real time (3). The experimental results and models which emerged from this study will be presented.

1. Uzgiris, E.E. & Kornberg, R.D., Nature 301, (1983) 125-129.
2. Brisson, A., Lambert, O. & Bergsma-Schutter, W. In: Crystallization of Nucleic Acids and Proteins: A practical Approach. (1998), Eds. Ducruix, A. & GiegE, R. (Oxford University Press), in press.
3. Reviakine, I., Bergsma-Schutter, W., Brisson, A. J.Struct.Biol. (1998), in press.

An unprocessed AFM image of a 2D crystal of a membrane-binding protein, annexin V, is shown (680x680 nm). Several stacking faults (green arrows) and a grain boundary (white arrow) are visible.