Advances in life sciences are inextricably linked to those in the technology of measurement. Understanding retinal proteins has also progressed by inventing new measurements and improving existing analytical methods. One of the earliest and still most powerful methods for studying retinal proteins is optical spectroscopic methods, such as UV-visible absorption spectroscopy [1], which allows to analyze photoreaction intermediates during functional processes of retinal proteins. Nuclear magnetic resonance (NMR) [2] and vibrational spectroscopy such as resonance Raman [3] and FTIR [4] provided detailed structural dynamics, while electron diffraction [5] and X-ray crystallography [6] visualized three-dimensional structures, and high-speed atomic force microscopy (HS-AFM) captured their motions [7]. In recent years, remarkable advances in cryo-electron microscopy [8] have allowed high-resolution structural analysis of rhodopsins in its membrane-embedded state, and X-ray free electron laser [9] monitors detailed structural dynamics in photointermediates. This session presented researches on advanced measurements and theoretical analytical methods that will deepen our understanding of retinal proteins. Four of the five talks were on experimental development and one on theoretical development.
The session was chaired by Uchihashi together with Kandori in place of Prof. Suneel Kateriya who could not come to the meeting.
The 1st speaker was Prof. Massimo Olivucci (University of Siena, Italy). He described a new quantum chemical model that can relate the amino acid sequence of retinal proteins to the brightness of their fluorescence and showed its usefulness in developing optogenetic tools [10].
The 2nd speaker was Prof. Eriko Nango (Tohoku University/Riken, Japan). She discussed the development of time-resolved serial femtosecond crystallography (TR-SFX) and pump-probe TR-SFX techniques and their application to Nonlabens marinius rhodopsin-3 [11] and bacteriorhodopsin.
The 3rd speaker was Prof. Mikihiro Shibata (Kanazawa University, Japan). He demonstrated the ability to rapidly analyze the oligomeric structure of microbial rhodopsin reconstituted in a lipid membrane using high-speed atomic force microscopy [12].
The 4th speaker was Dr. Kenichi Ataka (Freie Universität Berlin, Germany). He discussed translocon-unassisted folding process of various microbial rhodopsin during cell-free expression by using Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS) [13].
The last speaker was Prof. Przemyslaw Nogly (Jagiellonian University, Poland). He talked about time-resolved serial crystallography of a chloride-pumping rhodopsin throughout the transport cycle from 10 ps to 50 ms [14], and discussed on the molecular mechanisms.
References
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- [9] Neutze, R., Wouts, R., van der Spoel, D., Weckert, E., Hajdu, J. Potential for biomolecular imaging with femtosecond X-ray pulses. Nature 406, 752–757 (2000). https://doi.org/10.1038/35021099
- [10] Barneschi, L., Marsili, E., Pedraza-González, L., Padula, D., Vico, L. D., Kaliakin, D., et al. On the fluorescence enhancement of arch neuronal optogenetic reporters. Nat. Commun. 13, 6432 (2022). https://doi.org/10.1038/s41467-022-33993-4
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- [12] Shibata, M., Inoue, K., Ikeda, K., Konno, M., Singh, M., Kataoka, C., et al. Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy. Sci. Rep. 8, 8262 (2018). https://doi.org/10.1038/s41598-018-26606-y
- [13] Ataka, K., Drauschke, J., Stulberg, V., Koksch, B., Heberle, J. pH-induced insertion of pHLIP into a lipid bilayer: In-situ SEIRAS characterization of a folding intermediate at neutral pH. Biochem. Biophys. Acta Biomembr. 1864, 183873 (2022). https://doi.org/10.1016/j.bbamem.2022.183873
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