Proteins are the biomacromolecular motors of life. While earlier accounts stressed the lock-key concept of protein function, a more complete understanding needs to take into account motions and molecular flexibility as ingredients how specific functions can be performed. In particular, larger conformational changes are known to reflect allosteric changes from active to passive states of enzymes and chaperons. However, it is unknown how these large conformational motions emerge from the fast local motions driving structural fluctuations. This study used a unique combination of fluorescence methods, neutron scattering and molecular dynamics simulations to successfully address this dynamical characterization for the challenging case of a multi-subunit chaperon.

Figure 1: Nanosecond fluorescence correlation spectroscopy (nsFCS, a) together with neutron spin echo spectroscopy (NSE, b) evidence molecule-spanning  motions on time scales of 150 ns, which can be connected to principal motions from molecular dynamics simulations (c).  By this combination, a mechanistic understanding of the onset of allosteric motions before the point of no return (PNR, d) is possible, opening new opportunities for biophysical understanding.

We studied the nanosecond motions of the heat shock protein Hsp90 comprehesively with state-of-the-art characterization. First, we used methods based on Förster resonance energy transfer (FRET). Using nanosecond fluorescence spectroscopy (nsFCS), we evidenced a relaxation time around 150 ns. Using time-resolved single-molecule anisotropy, we excluded rotations as origin of this motion, thus suggesting more interesting large-scale structural fluctuations. Second, we could observe signatures of these molecule-spanning motions in neutron spin echo spectroscopy at IN15 (ILL), as an additional relaxation channel on top of translational and rotational diffusion. Third, the stuctural signature could be matched with a cartesian principal component analysis (PCA) of the molecular dynamics from microsecond-long atomistic simulations, outlining specific highly diffusive and fluctuating bending-contraction motions as molecular interpretation. Finally, we could show that the presence of a cochaperon shifted the nanosecond dynamics, suggesting that functional understanding is out there to explore.

The experimental methodology opens opportunities for a more mechanistic understanding of onset of allostery, which could have implications for a large number of systems in life science and pharmacy. Besides extending the characterization to new systems, the combination of fluorescence and quasi-elastic neutron scattering (QENS) is also promising to better understand how overall structural dynamics from QENS can be connected to specific local information from fluorescence techniques. We thus aim to also perform both careful validation and explorative comparisons to build a methodological bridge between the two communities, hopefully enabling fundamental understanding as well as future applications in biomedicine.

The study was performed in a multinational collaboration between scientists at the University of Freiburg (Germany), the Institut Laue Langevin (France), the University of Tübingen (Germany), Malmö University and Lund University (Sweden). We combined advanced experimental characteriyation using fluorescence methods and quasi-elastic neutron scattering with atomistic molecular dynamics simulations and a common interpretation and analysis frame.

Contact

Assoc. Prof.  Felix Roosen-Runge

Division of Physical Chemistry, Lund University
felix.roosen-runge@fkem1.lu.se