Rapid protein crystallization by surface mutagenesis

描述（申请人提供）：在过去的三十年里， 晶体学已经成为生物医学领域最有力的工具之一， 以理工科为重然而，尽管相关技术取得了巨大进步， 在分子生物学中，蛋白质结构的确定通常仍然是一个 这是一个挑战，可能需要数年才能完成。目前有两个战略 就业：研究侧重于一个特定的结构（S）选择，因为 已知的生物学作用;和结构基因组学，其中大量的 选择目标-不一定具有已知功能和折叠-以允许 对于高吞吐量结构确定和全局集合的采样 折叠模式。这两种方法都有缺点： 结晶学可能需要很长时间才能取得成果，而结构 基因组学策略将可能允许每5个中有1个的结构解决方案 10个尝试的蛋白质在这两种情况下，限制因素可能是 衍射质量晶体的制备。 在本申请中，我们建议调查的可行性， 通过“晶体工程”，即诱变蛋白质结晶 合理选择表面残留物。虽然诱变已被证明， 过去影响蛋白质的溶解度和结晶模式， 从未以理性的方式使用，作为一种技术也不可行 高通量筛选方法在任何规模的实验室。的方法 在我们的试点研究中进行的测试整合了三种方案：使用 Gateway方法，一种优化的QuikChange诱变方案， 具有高构象熵和高概率的残基的诱变 在表面上发生的（即赖氨酸和谷氨酸）。初步结果， 使用人蛋白质RhoGDI（Rho鸟嘌呤核苷酸解离抑制剂）， 来自PDZ-RhoGEF LH结构域和来自GRAF的BH结构域显示K2 A（Lys 和E2 A（Glu到Ala）单突变体和多突变体结晶得多 比野生型蛋白更容易。这与我们的假设一致， 表面构象熵产生结晶屏障。 从试验性实验中得出的初步结论表明， 高通量晶体工程可以显著地提高 晶体结构测定：我们表明，在两周内获得 可以筛选多达10种或更多种变体的结晶 选择的蛋白质。该方法简单易行，成本低廉 在任何规模的实验室里。要求提供资金，以进一步测试 该方法的适用性和成功率，并将该方法应用于 为结构基因组计划选择的目标子集。

DESCRIPTION (provided by applicant): In the past three decades, macromolecular crystallography has emerged as one of the most powerful tools in biomedical sciences. However, in spite of dramatic advances in relevant technologies and in molecular biology, structure determination of a protein often remains a challenge and may take years to complete. Two strategies are currently employed: research which focuses on a specific structure(s) chosen owing to the known biological role(s); and structural genomics, where large numbers of targets - not necessarily with known function and fold - are selected to allow for high throughput structure determination and sampling of the global ensemble of folding patterns. Both approaches suffer from shortcomings: targeted crystallography may take a long time before it bears fruits, while structural genomics strategies will probably allow for structure solution of 1 in every 5 to 10 attempted proteins. In both cases, the limiting factor is likely to be the preparation of diffraction-quality crystals. In this application we propose to investigate the feasibility of efficient protein crystallization through 'crystal engineering', i.e. mutagenesis of rationally selected surface residues. Although mutagenesis has been shown in the past to affect the solubility and crystallization modes of proteins, it was never used in a rational fashion, nor was it technically feasible as a high-throughput screening method in a laboratory of any size. The approach tested in our pilot study integrates three protocols: rapid cloning using the Gateway method, an optimized QuikChange mutagenesis protocol, and systematic mutagenesis of residues with high conformational entropy and high probability of occurring on the surface (i.e. Lys and Glu). Preliminary results, obtained with the human protein RhoGDI (Rho Guanine nucleotide Dissociation Inhibitor), the LH domain from PDZ-RhoGEF, and the BH domain from GRAF, show that K2A (Lys to Ala) and E2A (Glu to Ala) single and multiple mutants crystallize much more readily than wild-type protein. This is in agreement with our hypothesis that surface conformational entropy creates a barrier for crystallization. Preliminary conclusions inferred from the pilot experiments suggest that high-throughput crystal engineering may significantly enhance the efficiency of crystal structure determination: we show that within two weeks of obtaining cDNA it is possible to screen for crystallization of up to 10 or more variants of the selected protein. The approach is simple and inexpensive, and feasible in a laboratory of any size. Funds are requested to further test the general applicability and success rate of this method, and to apply the method to a subset of targets selected for the Structural Genomic Initiative.