MECHANISM OF THROMBIN RECEPTOR ACTIVATION DEACTIVATION

我的实验室对f-肌动蛋白的结构和功能感兴趣。 结合蛋白。我们正在不断地确定 核磁共振和核磁共振研究f-肌动蛋白结合蛋白的“头饰”结构域 X射线结晶学。我们已经表达了一系列突变体 在大肠杆菌中的头盔，需要进行质谱分析以确保 我们提纯的产品是正确的序列，并且没有 在提纯过程中进行修改。最近，我们发展了一些条件 在那里我们可以重复地得到衍射式头饰的水晶 在X射线束中(分辨率超过1.8A)。为了让 获得求解高分辨率X射线所需的位相信息 维林头饰的晶体结构，我们转向多重 异常离散法(MAD)。疯狂阶段化要求数据 在同步加速器源收集，头戴式导数标有 含硒蛋氨酸。布鲁克海文的数据收集时间 同步加速器源有限，必须申请。因此，它 必须准备好我们的硒标记晶体并 预先具有特征的。IS对我们来说很重要，要确保我们的 标记是有效的，此外，硒不是 在我们参观之前，在提纯过程中氧化或丢失 同步加速器。该中心进行的质谱分析是 对确保我们的晶体中含有预期的硒和 因此适合于同步加速器数据收集和MAD 阶段化。没有其他方法比质谱分析更有效的了 提供硒的存在和占有率的证明 在我们的蛋白质晶体中。

My lab is interested in the structure and function of f-actin binding proteins. We arecurrently determining the structure of the "headpiece" domain of the f-actin bundfing protein villin by NMR and X-ray crystallography. We have expressed a series of mutants of headpiece in E. coli and require mass spectral analysis to insure the product that we purify is the correct sequence and has not been modified during purification. Recently, we have developed conditions where we can reproducibly get crystals of headpiece that diffiract well in the X-ray beam (to beyond 1.8 A resolution). In order to the obtain phase information necessary to solve the high resolution X-ray crystal structure of villin headpiece, we turned to the multiple anomolous dispersion (MAD) method. MAD phasing requires data to be collected at a synchrotron source with headpiece derivatives labeled with selenomethionine. Data collection time at the Brookhaven Synchrotron Source is limited and must be applied for. Therefore, it was essential to have our selenium-labeled crystal prepared and characterized in advance. Is was important for us to insure that our labeling was effective and, in addition, that the selenium was not oxidized or lost during thepurification, before our visit to the synchrotron. The mass spectral analysis performed by the center was critical in insuring our crystal contained the expected selenium and was therefore suitable for synchrotron data collection and MAD phasing. No other method is as effective as mass spectral analysis in providing proof of the presence and percent occupancy of the selenium in our protein crystals.