Rapid protein crystallization by surface mutagenesis

描述(申请人提供)：在过去的三十年里， 结晶学已经成为生物医学中最强大的工具之一 科学。然而，尽管相关技术和技术取得了巨大进步， 在分子生物学中，蛋白质的结构确定通常保持在 挑战，可能需要数年时间才能完成。目前有两种策略 受雇：专注于特定结构的研究(S)，原因是 已知的生物学作用(S)；以及结构基因组学，其中大量 选择目标-不一定具有已知的功能和折叠-以允许 用于全球集合的高通量结构确定和采样 折叠式图案。这两种方法都有缺点：有针对性 结晶学可能需要很长时间才能结出硕果，而结构 基因组学战略可能会允许每5个人中就有1人的结构溶液 到10种尝试的蛋白质。在这两种情况下，限制因素很可能是 衍射级晶体的制备。 在这个应用中，我们建议研究高效的可行性 通过‘晶体工程’实现蛋白质结晶，即诱变 合理选择表面残留物。尽管诱变作用已经在 过去影响蛋白质的溶解度和结晶方式的是 从来没有以理性的方式使用过，在技术上也不可行 在任何规模的实验室中的高通量筛选方法。该方法 在我们的试点研究中测试了三种协议：使用 一种优化的QuikChange突变协议--网关方法和系统 高构象熵和高概率残基的诱变 发生在表面的(即赖氨酸和谷氨酸)。初步结果，获得 用人类蛋白RhoGDI(Rho鸟嘌呤核苷酸解离抑制物)， 来自PDZ-Rhogef的LH域和来自Graf的BH结构域表明K2a(Lys Ala)和E2a(Glu To Ala)的单个和多个突变体结晶得更多 比野生型蛋白质更容易。这与我们的假设是一致的 表面构象熵为结晶创造了障碍。 从试点实验中得出的初步结论表明， 高通量晶体工程可能会显著提高 晶体结构测定：我们证明在获得晶体结构的两周内 可以筛选多达10个或更多变体的结晶 所选蛋白质的。该方法简单易行，成本低廉。 在任何规模的实验室里。需要资金来进一步测试这位将军 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.