W0157: X-ray Diffraction from Proteins Crystallized in Polyacrylamide Gels

The feasibility of measuring the x-ray diffraction patterns from protein crystallites grown in polyacrylamide gels was investigated using the insertion device beamline and experiment facilities of the Structural Biology Center (Sector 19 at Argonne’s Advanced Photon Source (APS) facility). An adaptation of the method of two-dimensional electrophoresis in a polyacrylamide gel matrix, pioneered and perfected at Argonne, can provide the capability to purify small amounts of hundreds of different proteins. Coupling this technique with the development of combinatorial methods for protein crystallization within the purification matrix and the use of synchrotron x rays from a high-brilliance source such as the APS would provide a rapid tool for screening hundreds of proteins for optimal crystallization conditions. Polyacrylamide gels were cast as cylinders of approximately 7 mm in diameter and 5 mm in height. As expected, the virgin gels showed a single, very broad diffraction maximum which is essentially featureless, extends from approximately 2.0 to 4.0 Å and peaks at approximately 3.0 Å. This maximum, however, does not interfere with the ability to observe diffraction from protein crystallites. By comparison, diffraction from protein crystallites is extremely sharp and the lower order, strongest maxima are generally seen at resolutions lower than diffraction from the gel. Even when they do occur within the range of “background” from the gel, diffraction features from protein crystallites are easily discerned. The initial test case was hen egg-white lysozyme, which crystallizes in a tetragonal P4 ₃2₁2 structure with a = 78 Å and c = 39 Å. Lysozyme solutions in concentrations of 10, 15 and 20 mg/ml were added to individual gels, along with acetate buffer and NaCl solutions ranging in concentration from 0 to 14%. After 2 or 3 days at ambient temperature, small crystals were observed visually in the gels with high lysozyme and salt concentrations. All gels contained lysozyme crystallites, as evidenced by observable x-ray diffraction features, after about 5 weeks at ambient temperature. The gel with the lowest lysozyme concentration showed very weak diffraction to 5.5 Å and that with the highest lysozyme and salt concentrations exhibited diffraction features to better than 2.0 Å resolution.