ROTATIONAL DIFFUSION WITH 2 PHOTON FCS

我们使用单光子共聚焦和双光子荧光 在单分子水平上检测蛋白质反应的光谱学。 通过测量单个蛋白质的过程，我们不受 系综平均 因此，蛋白质的异质性程度， 并且可以监测可用的反应途径的多样性 直接. 初步实验进行的结合， 8-苯胺基-1-萘磺酸（ANS）转化为脱镁血红蛋白。 的 ANS的量子产率增加超过2个数量级， 配体与蛋白质结合，使其能够区分 蛋白质的连接和未连接构象之间的差异。 的 蛋白质被单独标记为绿色， 定位并固定在硅胶中。 ANS扩散通过 玻璃，以及随时间监测的结合事件。 率 然后可以从时间中提取反应的系数 绑定事件的顺序。

We are using one-photon confocal and two-photon fluorescence spectroscopy to detect protein reactions on the single molecule level. By measuring processes on single proteins, we are not subject to ensemble averaging. Thus, the degree of heterogeneity in proteins, and the variety of reaction pathways available can be monitored directly. Initial experiments are performed on the binding of 8-anilino-1 -naphthalene sulfonic acid (ANS) to apornyoglobin. The quantum yield of ANS increases by over 2 orders of magnitude when the ligand is bound to the protein, making it possible to distinguish between the ligated and unligated conformations of the protein. The proteins are singly labeled with oregon green to make them easy to locate and are immobilized in a silica gel. The ANS diffuses through the glass, and the binding events monitored with time. The rate coefficients of the reaction can then be extracted from the time sequence of binding events.