Abstract
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.
Highlights
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity
The experimental environment for X-ray free electron laser (XFEL) is different from that used for synchrotron-based crystallography which requires cryogenic cooling protection, and this difference could influence the interaction of ligands or ions that are related to membrane protein function
Studies have indicated that XFEL-derived structures do not suffer from perturbations because of cryogenic cooling or radiation damage from synchrotrons [34, 43]
Summary
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps. Non-cryogenic structure of ClR derived from XFEL protonated Schiff base (PSB) changes according to the unique residue of the proton acceptor and proton donor in rhodopsin, which induces a difference in the ion transport pathway and pumping mechanism. XFEL-derived structures for proton-pumping rhodopsins have been reported and compared with those determined at synchrotrons, and the proton-transporting mechanism was clearly identified from the nanosecond to millisecond time scale with retinal photoisomerization using timeresolved SFX experiments [21, 22, 34]. The structural information of ClRs close to the native condition (room temperature) and the correct ion-transporting mechanism using time-resolved SFX methods remain unknown.
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