Abstract
While in chapter 3 the effect of lipid composition on the function of bacteriorhodopsin was demonstrated, it is clear that the use of synthetic lipid in the constitution of nanodisc deviates from the native environment of membrane protein. In this chapter, an efficient method of direct extraction was presented. The method can extract bacteriorhodopsin directly from the native purple membrane and incorporate into nanodisc without the addition of synthetic lipid or extra lipid extracts. The transfer efficiency was found to be up to 38% higher, and was found that in the presence of high salt environment to maintain trimeric conformation that is native to the protein. The native membrane nanodisc was examined using high resolution Zernike phase TEM without staining. The lipid composition of the native membrane nanodisc was conducted using LC-ESI-MS and 31P NMR, which showed that the majority of essential phospholipid was successfully transferred to the nanodisc from native purple membrane. Finally, the preservation of protein function was investigated using transient absorption spectroscopy, and was found to be photocycle active suggesting the function of bacteriorhodopsin was maintained on the native membrane nanodisc.
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Acknowledgements
The Zernike phase TEM image was taken by Pai-Chia Kuo from the Institute of Physics of Academia Sinica, Taipei, Taiwan. Dr. Shing-Jong Huang from the Instrumentation Center of National Taiwan University, Taipei, Taiwan, performed the 31P NMR experiment. LC-ESI-MS experiment was operated by Dr. Chien-Hung Chen of Core Facilities in Genomic Research Centre, Academia Sinica, Taipei, Taiwan. Transient absorption spectroscopy was performed at National Tsing Hua University, Hsinchu, Taiwan, with the help of Tsung-Yen Lee.
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Yeh, V. (2019). Native Membrane Nanodisc. In: Study of Bacteriorhodopsin in a Controlled Lipid Environment. Springer Theses. Springer, Singapore. https://doi.org/10.1007/978-981-13-1238-0_5
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DOI: https://doi.org/10.1007/978-981-13-1238-0_5
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