W0155

X-ray crystallography is severely limited by the propensity of the protein targets to form suitable crystals. According to estimates obtained from high-throughput structural genomics studies, approximately 20% of soluble, purified proteins yield single crystals. Recently, we proposed that rational mutagenesis of residues with high conformation entropy (e.g. Lys and Glu), is likely to create epitopes facilitating crystallization. Studies with the model protein RhoGDI^1,2showed that this approach leads to enhanced propensity of the mutants to yield X-grade crystals, and that some of these crystals can exhibit superior quality, diffracting to atomic resolution. Single and multiple mutants were tested: Lys to Ala, Lys to Arg and Glu to Ala. Double and triple mutants targeting clusters of Glu/Lys work best. We also discovered that the mutated epitopes mediate crystal contacts in the predicted way, so that there is a causal relationship between the mutagenesis and the ability of the protein to crystallize. Next, we applied the strategy to three proteins recalcitrant to crystallization: the RGS domain of PDZRhoGEF, the Lcrf antigen of Yersinia pestis, and the N-terminal domain of doublecortin, the product of the causal gene for brain lissencephaly and the double-cortex syndrome. All three yielded X-grade crystals from an initial screen of up to 5 mutants.

Our present work concentrates on the application of these principles to twenty proteins selected by the Midwestern Center for Structural Genomics from the B. subtilis genome that failed to crystallize in the initial crystallization screen. Preliminary studies show that any evaluation of the success rate of our method must take into account the incidence of erroneous clones, implicit in high-throughput approach, as well as natively unfolded proteins, which may be identified by 2D NMR or CD spectroscopy. We have examples of both, as well as examples of proteins crystallized in the wild-type form and after surface mutagenesis.