Production of eukaryotic heliorhodopsins for structural analysis utilizing the LEXSY expression system

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Heliorhodopsins (HeRs) are a distinct class of microbial rhodopsins with inverted termini compared to Type I rhodopsins.
• Producing HeRs for research has been difficult, with limited structural and functional data available.
• No eukaryotic HeRs had been reported prior to this study.

Purpose of the Study:

• To report the first successful expression and characterization of eukaryotic heliorhodopsins.
• To demonstrate the functional state of these newly expressed HeRs.
• To enable future structural and functional investigations of eukaryotic HeRs.

Main Methods:

• Expression of three eukaryotic heliorhodopsins using the LEXSY system.
• Spectroscopic characterization to confirm functional states.
• Crystallization of the expressed eukaryotic heliorhodopsins.

Main Results:

• Successfully expressed three eukaryotic heliorhodopsins from diverse eukaryotic sources (marine/freshwater algae, marine unicellular eukaryote).
• Spectroscopic analysis confirmed that the expressed HeRs were in functional states.
• Achieved successful crystallization of the eukaryotic heliorhodopsins.

Conclusions:

• This study presents the first successful expression and crystallization of eukaryotic heliorhodopsins.
• The findings pave the way for detailed structural and functional studies of eukaryotic HeRs.
• This work expands the known diversity and accessibility of microbial rhodopsins for research.