Proj 1: Improving macromolecule crystallization using symmetry-inducing GFP

摘要/摘要 利用GFP晶格促进变异体改善大分子结晶 在X射线结晶学结构测定中的瓶颈是结晶，其中-70%的纯化蛋白质失效。蛋白质不能结晶的一个主要原因是没有合适的晶格形成接触。这个项目的中心假设是，每当测试一种具有新的潜在晶体接触的新形式的分子时，获得晶体的可能性就会增加。我们将开发快速和通用的方法，通过将蛋白质与一系列低聚形式的绿色荧光蛋白(GFP)联系起来，在蛋白质分子上产生大量脉络。使用GFP是一个主要的优势，因为它可以通过将发夹(由环连接的两条链)插入到目标蛋白质的环中而牢固地连接到几乎任何蛋白质上。含有这个额外发夹的目标蛋白可以紧密结合到缺失发夹的“分裂”版本的GFP上。这意味着我们可以预先创建一组单体和不同表面之间具有不同关系的Split-GFP低聚物。插入一个环中的带有GFP发夹的目标蛋白质或复合体可以单独纯化，然后与每个Split-GFP模块结合，而不需要任何进一步的遗传操作、蛋白质表达或成分纯化，从而产生大量可以结晶的不同复合体。或者，GFP模块本身(而不是发夹)可以在允许的循环和旋转下直接插入到目标蛋白支架中。GFP试剂和使用它们的方法将在一组模型蛋白质上进行验证。我们假设，这些包含不同齐聚形式的GFP支架试剂的可获得性将极大地扩大可能的晶格接触的范围，并显著改善蛋白质和蛋白质复合体的结晶性，否则可能难以结晶。这项工作将导致通过促进人类疾病中涉及的蛋白质的结构确定来增加我们对人类健康的理解和我们治愈人类疾病的能力的方法。

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.