MUTATIONAL APPROACHES TO INVESTIGATION OF PROTEIN STRUCTURE, FUNCTION & FOLDING

我的实验室对f-肌动蛋白的结构和功能很感兴趣 结合蛋白 我们正在确定 通过NMR测定f-肌动蛋白激活蛋白绒毛蛋白的“头部”结构域， X射线晶体学 我们已经表达了一系列的突变体， 在E.大肠杆菌，并需要质谱分析，以确保 我们纯化的产物是正确的序列， 在纯化过程中改性。 最近，我们已经创造了条件 在那里我们可以重复地得到头盔的晶体， 良好的X射线束（超过1.8 A的分辨率）。 以便于 获得解决高分辨率X射线所需的相位信息 晶体结构的绒毛头盔，我们转向了多个 反常色散（MAD）方法。 MAD阶段化要求数据 在同步加速器源处收集， 硒代蛋氨酸 Brookhaven的数据收集时间 同步辐射源数量有限，必须申请。 因此 是制备硒标记晶体的关键， 提前的特点。 对我们来说，确保我们的 标记是有效的，此外，硒不是 在我们参观之前，在净化过程中氧化或丢失， 同步加速器 该中心进行的质谱分析是 关键在于确保我们的晶体含有预期的硒， 因此，适用于同步加速器数据收集和MAD 分阶段 没有其他方法像质谱分析一样有效， 提供了硒的存在和百分比占据的证据 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.