Götz Markus, Wortmann Philipp, Schmid Sonja, Hugel Thorsten
Institute of Physical Chemistry, University of Freiburg.
Institute of Physical Chemistry, University of Freiburg; Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology.
J Vis Exp. 2018 Jan 30(131):56896. doi: 10.3791/56896.
Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.
单分子荧光共振能量转移（smFRET）已成为一种广泛应用的生物物理技术，用于研究生物分子的动力学。对于细胞中的许多分子机器而言，蛋白质必须在功能循环中与相互作用伙伴协同作用，以完成其任务。将双色smFRET扩展到多色smFRET，使得同时探测多个相互作用或构象变化成为可能。这不仅为smFRET实验增添了新的维度，还提供了独特的可能性，即当使用固定化样品和全内反射荧光显微镜（TIRFM）时，直接研究事件序列并检测相关相互作用。因此，多色smFRET是一种通用工具，可用于以定量方式且以前所未有的细节研究生物分子复合物。在此，我们展示了如何克服蛋白质多色smFRET实验中的特殊挑战。我们给出了获取数据和提取动力学信息的详细方案。这包括轨迹选择标准、状态分离，以及使用三维整体隐马尔可夫模型（HMM）从噪声数据中恢复状态轨迹。与其他方法相比，动力学信息不是从停留时间直方图中恢复，而是直接从HMM中恢复。最大似然框架使我们能够严格评估动力学模型，并为速率提供有意义的不确定性。通过将我们的方法应用于热休克蛋白90（Hsp90），我们能够解开该蛋白的核苷酸结合和全局构象变化。这使我们能够直接观察Hsp90二聚体两个核苷酸结合口袋之间的协同作用。
