Structural Dynamics of Hsp90 Resolved by a Novel Multi-Pair FRET Approach

Three-dimensional models of proteins at atomic resolution are an essential prerequisite for mechanistic insight into protein function. However, in solution the protein exists as a dynamic equilibrium of global and local conformations. Therefore, a comprehensive understanding of protein function requires knowledge on the structure - dynamics relationship. We developed a novel hybrid approach based on single molecule Forster Resonance Energy Transfer [1, 2] that integrates hundreds of time resolved intramolecular distances together with x-ray structure information and MD simulations. Simultaneous determination of fluctuations and time resolved anisotropies enables a new level of accuracy and confidence. We demonstrate the power of our method by determining the dynamic structure of the molecular chaperone and heat shock protein Hsp90. Our resulting mean structure for the closed state of Hsp90 resembles the corresponding x-ray structure of yeast Hsp90 [3] to 2.5 A. Beyond that, we resolved the previously unknown open structure of this multi-domain protein. Surprisingly, the open state shows much larger fluctuations on relevant timescales than the closed state. Finally, we show how this method can be applied to quantify inter-domain dynamics and dynamics of small elements. We anticipate that this dynamic structure determination will solve many long standing questions regarding flexible parts in large proteins and protein-protein interactions.[1] Muschielok A. et al.; Nature Methods 2008; 5, 965-971.[2] Kalinin S. et al.; Nature Methods 2012; 9(12):1218-1225.[3] Ali M.M. et al.; Nature 2006, 440(7087):1013-1017.