Preparing high resolution membrane protein crystals

描述（由申请人提供）：膜蛋白的X射线晶体学，其中许多是重要的药物靶点，是一个具有挑战性和至关重要的研究领域。不幸的是，它明显落后于细胞溶质蛋白的X射线衍射研究。有两个具体的技术问题，占这一点：缺乏有效的表达系统的膜蛋白（特别是那些真核生物起源）和困难的制备单晶适合X射线衍射。使用当前技术获得的晶体通常质量差，并且通常在3.0 - 5.0 A的范围内结晶，产生靶蛋白质的相对不精确的原子模型。 由主要研究者进行的研究确定，可溶性蛋白质的合理表面诱变可以是一种强大的方法，允许制备蛋白质晶体，否则将无法进行结构研究，并且在某些情况下，可以获得比野生型晶体衍射分辨率高得多的新型晶体形式。该方法，基于表面熵减少（SER）的概念，是第一次成功的尝试，旨在合理诱导结晶工程表位介导晶体接触，从而导致成核和生长。迄今为止，已经使用这种方法获得了许多新蛋白质的晶体。我们现在建议使用类似的原理进行可行性研究，通过工程化更好的晶体接触来提高膜蛋白晶体的质量。为此，我们将使用两种蛋白质作为模型系统：E。coil机械敏感和电压调制通道（MscS）;大肠杆菌水通道蛋白AqpZ。这些蛋白质可以相对高的产量过表达，使它们特别适合我们的目的。MscS公布的结构被限制在3.9 A分辨率，而AqpZ的几种晶体形式都没有产生X射线数据来进行分辨率验证结构研究。我们的试点研究，在R21提案的高风险/高影响的框架内，将旨在建立一个长期的研究合理的表面诱变膜蛋白的高分辨率晶体学研究的可行性。

DESCRIPTION (provided by applicant): X-ray crystallography of membrane proteins, many of which are important drug targets, is a challenging and critically important field of research. Unfortunately, it lags significantly behind x-ray diffraction studies of cytosolic proteins. There are two specific technical problems that account for this: lack of efficient expression systems for membrane proteins (especially those of eukaryotic origin) and difficulties in the preparation of single crystals suitable for x-ray diffraction. The crystals that are obtained using current techniques are often of poor quality and typically diffract in the range of 3.0 - 5.0 A, yielding relatively imprecise atomic models of the target proteins. Studies conducted by the Principal Investigator established that rational surface mutagenesis of soluble proteins can be a powerful method allowing for the preparation of crystals of proteins otherwise recalcitrant to structural studies, and in some cases to obtain novel crystal forms diffracting to much higher resolution than wild-type crystals. The approach, based on the concept of surface entropy reduction (SER), is the first successful attempt designed to rationally induce crystallization by engineering epitopes that mediate crystal contacts and consequently lead to nucleation and growth. To date, a number of crystals of new proteins have been obtained using this method. We now propose to use similar principles to conduct a feasibility study of improving the quality of membrane protein crystals by engineering better crystal contacts. To that end, we will use two proteins as model systems: the E. coil mechanosensitive and voltage modulated channel (MscS), and the E. coli aquaporin AqpZ. These proteins can be overexpressed with relatively high yields, making them particularly suitable for our purposes. The MscS published structure was limited to 3.9 A resolution, while none of the several crystals forms of AqpZ yielded X-ray data to a resolution warranting structural investigation. Our pilot study, within the high risk/high impact framework of an R21 proposal, will aim to establish the feasibility of a longer-term research into rational surface mutagenesis of membrane proteins for high-resolution crystallographic studies.