As we know from the lock-and-key principle, the function of biomolecules depends strongly on their structure. The whole story becomes even more complex when we consider that the structure and functionality of a single biomolecule depends on its environment, the living cell. A complicated web of dependencies that is difficult to untangle in an experiment. A UniSysCat team involving the groups of Joachim Heberle and Ramona Schlesinger was now able to investigate a microbial chloride pump, which is of interest for medical applications, in living cells using time-resolved IR and UV/Vis spectroscopy.
Ion pumps are proteins which regulate the transport of vital ions from one side of a cell membrane to the other. Chloride pumps for example are membrane proteins that can pump chloride ions against a concentration gradient. Special ion pumps that are stimulated by light to start their pumping function are very interesting for medical applications, particularly optogenetics. For the development of new optogenetic tools, it is important to understand the underlying molecules and processes in detail - especially how they take place in living organisms.
The UniSysCat team has now taken a closer look at the function of one such ion pump, namely the halorhodopsin NmHR, a chloride pump found in a marine bacterium. Using time-resolved IR and UV/Vis spectroscopy, they were able to observe how the ion pump changes its structure in response to the irradiation with light.
With their approach, the team was able to monitor transient structural changes of the retinal cofactor, which is the part of the chloride pump that mainly controls the response to irradiation. Moreover, out of the many signals observed during the response of the chloride pump, they could clearly resolve the deprotonation of a single cysteine residue which is suspected to play a special role in the pumping process. These results show that time-resolved UV/Vis and IR spectroscopy are indeed useful tools to visualize the smallest structural changes in whole proteins, even in living cells.
The methodological approach presented in the study is therefore an excellent tool for investigating molecular processes at the level of individual amino acid residues in the environment of whole cells with high spatial and temporal resolution. This development gives a new direction to the field of in-cell studies and optogenetics. Moreover, the results are also groundbreaking for future UniSysCat projects when it comes to understanding catalytic processes in living cells.
This study has been published in JACS: Sabine Oldemeyer, Mariafrancesca La Greca, Pit Langner, Karoline-Luisa Lê Công, Ramona Schlesinger, and Joachim Heberle, Journal of the American Chemical Society 2024 146 (28), 19118-19127, https://doi.org/10.1021/jacs.4c03891