Long-term tracking of protein conformational dynamics through single-molecule thermal snapshot spectroscopy
Final Report Abstract
Overcoming the technical problems required to set up this new thermal snapshot experiment proved to be significantly more challenging and time-consuming than anticipated. By using fluorescent labels attached to myoglobin as sensors for the presence or absence of a bound CO ligand, we obtained a test system for investigating fluctuations in protein reactions over time that is generally well understood and thus provides a solid basis for initial experiments. At the same time, since the label reports directly on the reaction rates of ligands, we gain direct insight into fluctuations of the functionally relevant internal processes of this protein, making this protein an interesting research focus in its own right. It is generally inferred from bulk experiments that the rebinding rates of CO after dissociation vary significantly between individual proteins. These experiments will show this directly on a single molecule basis, as well as allow us to investigate these processes in much more detail. Already, one interesting result of our experiments is that even at room temperature, CO will likely rebind immediately to the protein after bond breaking, rather than the complex dissociating.
Publications
- “Microbial Halorhodopsins: Light- Driven Chloride Pumps”; Chemical Reviews (2018), 118(21), 10629-10645
Engelhard, C; Chizhov, I; Siebert, F; Engelhard, M
(See online at https://doi.org/10.1021/acs.chemrev.7b00715) - “Single electron transfer events and dynamical heterogeneity in the small protein azurin from Pseudomonas aeruginosa”; Chemical Science (2020), 11(3), 763-771
Pradhan, B; Engelhard, C; Van Mulken, S; Miao, XY; Canters, GW; Orrit, M
(See online at https://doi.org/10.1039/C9SC05405G)
