GROWING PROTEIN CRYSTALS BY HOOK OR BY CROOK: FROM MOLECULAR BIOLOGY TO LIPIDS. Aled M. Edwards, Cancer Research Group, Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada.

We have used two general approaches to facilitate the growth of protein crystals for X-ray diffraction experiments. The first approach is called SAF, for smallest, active fragment. SAF is simply a logical extension of the old observation that proteolytic fragments of proteins often both harbour complete biological activity and form useful crystals more readily than the full length protein. However, rather than depend on the fortuitous position of protease recognition sites, SAF couples partial proteolysis with deletion analysis in order to refine the boundaries of the domain of interest. In practice, an iterative cycle of partial proteolysis, deletion analysis, biochemical assays, protein over-expression and crystallization is used to identify a domain that forms diffraction-quality crystals. SAF capitalizes on the ease and rapidity of subcloning, over-expressing and purifying proteins as well as the technical ease and availability of N-terminal sequencing and mass spectrometry. We have used the SAF approach for three proteins, and we have generated fragments of each that were suitable for structure determination.

The second approach. lipid-layer seeding, uses lipid monolayers to seed crystal growth. We observed several years ago that two-dimensional protein crystals grown on lipid layers effectively nucleate the epitaxial growth of three dimensional protein crystals. In this way, crystals suitable for X-ray crystallography can be grown more rapidly, and at substantially lower protein concentrations and precipitants than for conventional crystal trials. The methodology has now been developed such that the procedure takes only a few seconds per crystal trial. We are now applying the method to an assortment of proteins in an effort to demonstrate the generality of the approach.