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Published on: August 31, 2012
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Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids
Ayumi Yamamoto1, Takashi Tsukamoto2, Kenshiro Suzuki1
1Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Japan.
Biophysical Journal
|May 13, 2020
Summary
This study successfully incorporated Natronomonas pharaonis halorhodopsin (NpHR) trimers into nanodisks (NDs), enhancing chloride binding and uptake kinetics. These findings highlight the importance of lipid interactions and trimer associations for efficient chloride pumping by NpHR.
Area of Science:
- Membrane biophysics
- Protein biochemistry
- Spectroscopy
Background:
- Halorhodopsins are light-driven chloride pumps crucial for cellular homeostasis.
- Reconstituting membrane proteins into nanodisks (NDs) offers a platform to study their function in a controlled environment.
- Understanding the factors influencing NpHR's chloride transport efficiency is key to its biotechnological applications.
Purpose of the Study:
- To reconstitute single Natronomonas pharaonis halorhodopsin (NpHR) trimers into nanodisks (NDs) using native archaeal lipids (NL) and POPC.
- To characterize the chloride binding affinity and photoreaction kinetics of ND-reconstituted NpHR.
- To investigate the impact of lipid environment and trimer interactions on NpHR function.
Main Methods:
- Nanodisk reconstitution of NpHR trimers.
- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for incorporation confirmation.
- Size-exclusion chromatography (SEC) for purity assessment.
- Visible circular dichroism (CD) spectroscopy for structural integrity.
- Absorption spectroscopy for chloride binding affinity (Kd) determination.
- Kinetic analysis of photoreaction cycles.
Main Results:
- Single NpHR trimers were successfully incorporated into NL-NDs and POPC-NDs.
- ND-reconstituted NpHR exhibited over 10-fold higher Cl--releasing affinity compared to native membrane fragments (MF NpHR).
- ND-reconstituted NpHR showed faster Cl- uptake kinetics than MF NpHR.
- POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting regulation by membrane surface charge.
- NL-ND NpHR showed altered Cl- binding and faster recovery, potentially due to reduced chromophore interactions.
Conclusions:
- Nanodisk reconstitution significantly enhances NpHR's chloride binding and uptake efficiency.
- The lipid environment, including headgroup charge and specific lipid-protein interactions (e.g., with bacterioruberin), modulates NpHR photocycle kinetics.
- Interactions between NpHR trimers and membrane structural flexibility are crucial for optimal chloride pumping activity.
