Proj 1: Improving macromolecule crystallization using symmetry-inducing GFP

总结/摘要 使用GFP的晶格促进变体改善大分子结晶 通过X射线晶体学进行结构测定的瓶颈是结晶，其中约70%的纯化蛋白质失败。蛋白质不能结晶的一个主要原因是没有足够的合适的晶格形成接触。该项目的中心假设是，每次测试具有新的潜在晶体接触的分子的新形式时，获得晶体的概率都会增加。我们将开发快速和通用的方法，通过将蛋白质连接到一系列低聚形式的绿色荧光蛋白（GFP）上，在蛋白质分子上产生许多脉络。GFP的使用是一个主要的优点，因为它可以通过将来自GFP的发夹（由环连接的两条链）插入靶蛋白的环中而与几乎任何蛋白质牢固地连接。含有这个额外发夹的靶蛋白可以与缺少发夹的GFP的“分裂”版本紧密结合。这意味着我们可以预先产生一组在单体和不同表面之间具有不同关系的分裂GFP寡聚体。携带插入一个环中的GFP发夹的靶蛋白或复合物可以单独纯化，然后与每个分裂GFP模块组合，而无需任何进一步的遗传操作、蛋白表达或组分纯化。以产生大量可结晶的不同络合物。或者，GFP模块本身（而不是发夹）可以在允许的环和转角处直接插入靶蛋白支架中。GFP试剂和使用它们的方法将在多组模型蛋白上进行验证。我们假设，这些体现不同寡聚体形式的GFP支架试剂的可用性将极大地扩展可能的晶格接触的库，并大大提高蛋白质和蛋白质复合物的结晶性，否则可能难以结晶。这项工作将导致方法，将增加我们对人类健康的理解和我们的能力，通过促进与人类疾病有关的蛋白质的结构确定来治愈人类疾病。

Summary/Abstract Improving macromolecular crystallization using lattice-promoting variants of GFP The bottleneck in structure determination by X-ray crystallography is crystallization, where -70% of purified proteins fail. A major reason proteins fail to crystallize is insufficient suitable lattice-forming contacts. The central hypothesis in this project is that the probability of obtaining crystals is increased every time a new form of a molecule with new potential crystal contacts is tested. We will develop rapid and general methods to generate numerous venations on a protein molecule by linking the protein to a series of oligomeric forms of green fluorescent protein (GFP). The use of GFP is a major advantage because it can be linked firmly to practically any protein by inserting a hairpin (two strands connected by a loop) from GFP into a loop of the target protein. The target protein containing this extra hairpin can bind tightly to a "split" version of GFP that is missing the hairpin. This means that we can create, in advance, a set of split-GFP oligomers with varying relationships between the monomers and varying surfaces^ The target protein or complex bearing the GFP hairpin inserted into one loop can be purified separately and then combined with each of the split-GFP modules, without any further genetic manipulation, protein expression, or purification of components, to yield a large number of different complexes that can be crystallized. Alternatively, the GFP modules themselves (rather than the hairpin) can be directly inserted into the target protein scaffold at permissive loops and turns. The GFP reagents and methods to use them will be validated on sets of model proteins. We hypothesize that the availability of these GFP scaffold reagents embodying different oligomeric forms will vastly expand the repertoire of possible lattice contacts, and substantially improve the crystallizability of proteins and protein complexes that may be otherwise difficult to crystallize. This work will lead to methods that will increase our understanding of human health and our ability to cure human illness by facilitating the structural determination of proteins involved in human disease.