Probing heterogeneity in the microbial rhodopsin–carotenoid complex

When light-sensitive proteins called microbial rhodopsins absorb light energy through retinal chromophores, this light absorption triggers a sequence of structural changes known as a photocycle. These changes underlie diverse biological functions, such as ion transport and light sensing; the engineered counterparts of these photoreceptive proteins are especially valuable for optogenetic experiments.

In some microbial rhodopsins, orangish pigments known as carotenoids function as light-harvesting antennas for the retinal chromophore. While using resonance Raman spectroscopy to probe the Gloeobacter rhodopsin–canthaxanthin (GR–CAN) complex — a model for studying microbial rhodopsin–carotenoid complexes — Kawano et al. made an unexpected observation: When they changed the excitation wavelength used for resonance Raman measurements, the Raman peak positions of retinal and canthaxanthin shifted continuously, suggesting that different excitation wavelengths selectively highlight slightly different molecular structures in the complex.

The team systematically investigated the excitation-wavelength dependence of the Raman shifts of the retinal and carotenoid chromophores of GR–CAN. The observed shifts revealed structural heterogeneity in both chromophores, with different excitation wavelengths selectively probing slightly different molecular structures within the complex. The Raman spectra also showed that the binding of one chromophore influences the electronic structure of the other.

“Our work shows that changing the excitation wavelength in resonance Raman spectroscopy can provide information about such hidden heterogeneity around a chromophore,” said author Yasuhisa Mizutani. “We hope this approach will become a useful way to investigate structural distributions in other light-sensitive and chromophore-containing proteins, and to connect this molecular heterogeneity with biological function.”

To understand the molecular origin of this heterogeneity, the team plans to identify the specific structural factors responsible for variations in the wavelength of maximum absorption.

“By examining how other Raman marker bands that report on different aspects of chromophore structure depend on the excitation wavelength, we hope to determine which structural variations underlie the observed differences in light absorption,” Mizutani said.

Source: “Structural inhomogeneity and inter-chromophore electronic/protein-mediated coupling in the Gloeobacter Rhodopsin–Canthaxanthin complex: Insights from resonance raman spectroscopy,” by Mizuki Kawano, Taito Urui, and Yasuhisa Mizutani, Journal of Chemical Physics (2026). The article can be accessed at https://doi.org/10.1063/5.0350151 .

This paper is part of the Festschrift in Honor of Tahei Tahara: Structure and Dynamics in Complex Molecular Systems Collection, learn more here .