Deciphering the mechanism of the human proteasome by mass photometry
Final Report Abstract
The ubiquitin-proteasome-system (UPS) is a vital machinery in eukaryotic cells which removes excess and defective proteins from the cell lumen. Proteins are targeted for destruction by attachment of a poly-ubiquitin sequence which triggers recognition by the proteasome. Malfunction of the UPS is typically associated with several (neurodegenerative) diseases. Despite recent advances in the understanding of the catalytic process based on single-molecules studies, a detailed picture of the of substrate binding and proteolysis is missing. We proposed to employ mass photometry (MP) to enhance the understanding of the involved processes on a single-molecule level. MP is a method based on interferometric detection of light scattered by single-protein complexes capable of inferring their molecular weight. We started with studying purified human 26s proteasome samples by MP. The results suggest that further improvements of the protein preparation and the experimental scheme were necessary. We established a novel combination of MP and single-molecule fluorescence microscopy. Analysis of the fluorescence intensity of individual MP events leveraged a substantial improvement of the mass resolution beyond established methods. Utilising the additional localisation information introduced by the simultaneous fluorescence detection we increased the sensitivity of MP compared to regular MP schemes. Furthermore, we implemented a holographic approach to MP based on total internal reflection illumination. We achieved optical holographic data of single protein molecules for the first time. Holographic data, in contrast to standard MP data, contains phase information which allows to determine the distance of molecules to the coverslip. Finally, we implemented a novel approach to efficiently perform MP on transmembrane proteins (TMP). Reconstitution of TMPs in peptidiscs showed MP performance superior to previously employed solubilisation methods like nanodiscs or detergent micelles. Although we deviated from the original main objectives, we made important progress on MP instrumentation which will allow future studies to engage with the UPS.
Publications
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Combination of single-molecule fluorescence and mass photometry. Biophysical Journal, 121(3), 430a-431a.
Pfitzner, Emanuel; Cole, Daniel & Kukura, Philipp
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Roadmap on Label‐Free Super‐Resolution Imaging. Laser & Photonics Reviews, 17(12).
Astratov, Vasily N.; Sahel, Yair Ben; Eldar, Yonina C.; Huang, Luzhe; Ozcan, Aydogan; Zheludev, Nikolay; Zhao, Junxiang; Burns, Zachary; Liu, Zhaowei; Narimanov, Evgenii; Goswami, Neha; Popescu, Gabriel; Pfitzner, Emanuel; Kukura, Philipp; Hsiao, Yi‐Teng; Hsieh, Chia‐Lung; Abbey, Brian; Diaspro, Alberto; LeGratiet, Aymeric ... & Lecler, Sylvain
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Single-protein optical holography.
Thiele, Jan Christoph; Pfitzner, Emanuel & Kukura, Philipp
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Characterization of membrane protein interactions by peptidisc-mediated mass photometry. iScience, 27(2), 108785.
Young, John William; Pfitzner, Emanuel; van Wee, Raman; Kirschbaum, Carla; Kukura, Philipp & Robinson, Carol V.
