Professor Thorsten Hugel
PhD Candidate Leonie Vollmar
Institute of Physical Chemistry II, University of Freiburg, Germany
The lab of Prof. Hugel is interested in obtaining real-time measurements of multi-protein complexes and their dynamic interaction. In living organisms, multi-protein complexes form intricate protein machines that regulate cellular processes. Of particular interest is the heat shock protein Hsp90 machinery, which consists of an Hsp90 dimer, co-chaperones, and client proteins. The complexes likely exist in multiple states that dynamically interchange and might be coordinated by utilizing the energy of ATP hydrolysis.
In the past, multicolor single-molecule FRET (Förster Resonance Energy Transfer) allowed Prof. Hugel and his team to study the Hsp90 machinery, determining the success of association and dissociation steps as well as large conformational changes of protein complexes simultaneously in real-time. Single-molecule FRET was also used to determine specific protein interfaces that interact as part of a complex. These studies impact the understanding of the Hsp90 machinery as well as general principles of multi-component protein systems, which is the basis for understanding cellular processes.
Studying protein dynamics in real-time requires the ability to image on a wide range of timescales, meaning that any increase in imaging bandwidth would help to obtain a better understanding of protein interactions across all lengths of experiment.
Further challenges were outlined by Prof. Hugel: “For a proper dynamic analysis of several states of protein interaction many single-molecule FRET traces are needed, therefore an increased field of view (FOV) would help. As the analysis is carried out on a pixel-by-pixel basis a very high degree of stability, reliability and reproducibility are required, this can only be achieved by perfect noise characteristics of the imaging device”.
With a sensor diagonal of 18.6mm, the Prime 95B improved the FOV by a factor of >2.5x allowing for more data to be acquired at the same time
- S. Schmid, M. Götz, T. Hugel (2016) Single-molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium, Biophysical Journal 111, 1375-84