Kinetics of protein complexes and their regulation across several time and length scales
Prof. Thorsten Hugel (Universität Freiburg)
Datum: 08.05.2020 um 15:30 Uhr
Ort: Webinar (Zoom)
Protein function is regulated on several length scales in living cells. While post-translational modifications act very locally, specific protein-protein interaction typically affect larger domains, and global changes (like crowding) affect the whole protein (complex) non-specifically.
Here, we show protein regulation under different degrees of localization, and present the effects on the heat shock protein 90 (Hsp90) chaperone system. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We will introduce a new mechanistic concept, namely functional stimulation through conformational confinement [1].
In a second part we use several single-molecule fluorescence based methods in combination with MD simulations to delineate the timescales for information transfer from the hydrolysis event in the nucleotide binding site of Hsp90 to large conformational changes in the Hsp90 dimer. This allosteric process occurs via a hierarchical mechanism that involves timescales from pico- to microseconds [2].
Finally, we demonstrate how the dynamics of heterogeneous low-affinity protein complexes can be determined by fast microfluidic mixing. Our mixing devices enables direct measurement of dissociation rates at a broad range of timescales from a few milliseconds to several minutes. We show the function of this device by measuring the dissociation rates and equilibrium dissociation constants of Hsp90 dimers [3] as well as Hsp90 and a co-chaperone and Hsp90 and a model substrate.
Altogether, these applications demonstrate that single-molecule fluorescence is becoming more and more versatile to investigate molecular mechanisms in multi-component (protein) systems across a wide range of timescales.
[1] S. Schmid and T. Hugel, Same Equilibrium. Different Kinetics. Protein Functional Consequences. BioRxiv, 838938 (2019); doi.org/10.1101/838938
[2] S. Wolf et al., Allosteric action of nucleotides on Hsp90 across several time- and length scales. BioRxiv, 2020.02.15.950725 (2020); doi.org/10.1101/2020.02.15.950725
[3] B. Hellenkamp et al., Kinetics of Transient Protein Complexes Determined via Diffusion-Independent Microfluidic Mixing and Fluorescence Stoichiometry. J. Phys. Chem. B, 122 (49), 11554 (2018).
Registration URL: https://lmu-munich.zoom.us/meeting/register/tJYsduisqzIpG9ZozJcR9irCfbKgiky-ji1m