Rocking movement in the anti-stress protein Hsp90
Proteins are the motors of the cell: They transport, among other things, nutrients, move our muscles, convert substances chemically or fold other proteins. The so-called heat shock protein Hsp90 is eminently important for our cells since it plays a decisive role in many basic processes – in humans as well as in bacteria or yeasts. For example, it is decisive in folding polypeptide chains into functioning proteins with very precisely defined spatial structures. Especially when cells are exposed to stress through heat or poisonous substances, Hsp90 production increases to keep the damage in check. The anti-stress protein is a dimer (which consists of two identical proteins) and can be roughly divided into three segments: the N terminal domain at the top, the middle domain and the C terminal at the bottom. Hsp90 taps the energy it requires for its work from the slow splitting of ATP, the fuel of every cell. In this process, the two strands move in opposing directions, albeit only a few nanometers. Some time ago we were able to observe this scissor-like N terminal movement in realtime. Our recent experiments show that the familiar one-ended scissor movement at the N terminal domains has to be extended to a rocking movement at both ends of the protein.Hsp90 opens and closes in a scissors-like manner at the C terminal as well – something hitherto unknown in dimers. We used the so-called FRET technology (FRET = Förster Resonance Energy Transfer) by attaching two fluorescent molecules at precisely defined positions in the Hsp90 and using these as a molecular ruler: When one pigment is illuminated, the other glows with increasing intensity the closer the two pigments get to each other. Using this effect, we were able to observe the nanometer-scale, double-ended scissor movement in individual Hsp90 dimers. Particularly interesting is that the double scissor movements at the N and C terminals are closely coupled: The Hsp90 dimer obviously opens and closes in alternation at each end, like a rocker. Surprisingly ATP bound at the N terminal domains regulates the motion at the C terminal end. Thus Hsp90 has to communicate internally across an unusually long distance of almost ten nanometers.
The Hsp90 molecules (size 5-10 nm) were caged in lipid vesicles with a diameter of about 200 nm (not to scale). The vesicles were immobilized via biotinylated lipids onto a solid substrate in a micro fluidic chamber and mounted in a prism-type TIR microscope. Single molecule fluorescence from the donor and acceptor were detected simultaneously by an EMCCD camera (A). Matching time traces were overlaid (B) and FRET efficiencies determined (C). The cumulated histogram of all FRET efficiencies shows two states that are clearly separated by a threshold (D), which allow to determine the rate constants from a separation of the time trace into open and close states (E).
"Dynamics of heat shock protein 90 C-terminal dimerization is an important part of its conformational cycle"
C. Ratzke, M. Mickler, B. Hellenkamp, J. Buchner and T. Hugel
PNAS, Online Early Edition in the week of August 23, 2010
PhD student in the group of
Prof. Thorsten Hugel, TU Munich
2006 - 2008
Master of Science in Biochemistry, TU Munich
2005 - 2006
Bachelor of Science in Biochemistry, TU München
2003 - 2005
Grundstudium in Biochemistry, Universität Regensburg
M. Mickler, M. Hessling, C. Ratzke, J. Buchner & T. Hugel:
The large conformational changes of Hsp90 are only weakly coupled to ATP hydrolysis
Nature Structural & Molecular Biology 16, 281 - 286 (2009)