Energy conversion of E. coli Hsp90 and yeast Hsp90

分子伴侣Hsp90参与多种底物蛋白的折叠、活化和稳定。其功能依赖于ATP结合和ATP水解。最近发表的热休克蛋白90的晶体结构显示了一个非常好的静态图片，并建议在ATP酶循环大的构象变化。我们开发了一种测定方法，允许在接近生理条件下真实的时间内跟踪单个Hsp90二聚体的构象动力学。这是用单分子荧光共振能量转移（smFRET）和特异性标记的单半胱氨酸突变体完成的，所述突变体特异性地固定在微流体室中。第一个实验使我们能够以核苷酸依赖的方式观察单个Hsp90分子在真实的时间内的大的构象变化。所获得的停留时间分布使我们能够提出一个动力学周期的开放和关闭的热休克蛋白90没有基板和共分子伴侣。该项目的目的是首先在各种（荧光）ATP类似物的帮助下将这些构象变化与Hsp90的化学循环联系起来。然后，底物和共分子伴侣结合将与热休克蛋白90的构象循环相关。最后，我们将对影响Hsp90活性的肿瘤药物的功能有新的认识。

The molecular chaperone Hsp90 is involved in folding, activation and stabilization of various substrate proteins. Its function is dependent on ATP binding and ATP hydrolysis. Recently published crystal structures of Hsp90 show a very good static picture and suggest large conformational changes during the ATPase cycle. We developed an assay that allows following the conformational dynamics of single Hsp90 dimers in real time under near physiological conditions. This is done with single molecule fluorescence resonance energy transfer (smFRET) and specifically labelled single-cystein mutants, which are specifically immobilized in microfluidic chambers. First experiments allowed us to observe the large conformational changes of single Hsp90 molecules in real time in a nucleotide dependent manner. The obtained dwell time distributions enabled us to propose a kinetic cycle for the opening and closing of Hsp90 without substrate and co-chaperones. The aim of this project is first to link these conformational changes to the chemical cycle of Hsp90 with the help of various (fluorescent) ATP analogues. Then, substrate and co-chaperone binding will be correlated with the conformational cycle of Hsp90. Finally, we will gain new insight into the function of tumor drugs that influence Hsp90 activity.