TY - JOUR
T1 - Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI
AU - Ueta, Tetsuya
AU - Kojima, Keiichi
AU - Hino, Tomoya
AU - Shibata, Mikihiro
AU - Nagano, Shingo
AU - Sudo, Yuki
N1 - Funding Information:
This work was financially supported by JSPS KAKENHI grant numbers JP19K16090 to K.K., JP18K06156 to T.H., JP18H01836 , JP19H05257 to M.S., and JP18H02411 , JP19H04727 , and JP19H05396 to Y.S. This research was partially supported by CREST-JST ( JPMJCR1656 ) and AMED ( 18dm0207060h0002 ) to Y.S.
Publisher Copyright:
© 2020 Biophysical Society
PY - 2020/11/3
Y1 - 2020/11/3
N2 - The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as “micelles.” The extracted protein is purified by column chromatography and then is often reconstituted into “liposomes” by removal of the detergent. The styrene-maleic acid (“SMA”) copolymer spontaneously forms nanostructures containing lipids without detergent. In this study, we applied SMA to characterize two microbial rhodopsins, a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin (RxR), and an unstable one, Halobacterium salinarum sensory rhodopsin I (HsSRI), and evaluated their physicochemical properties in SMA lipid particles compared with rhodopsins in micelles and in liposomes. Those two rhodopsins were produced in Escherichia coli cells and were successfully extracted from the membrane by the addition of SMA (5 w/v %) without losing their visible color. Analysis by dynamic light scattering revealed that RxR in SMA lipid particles (RxR-SMA) formed a discoidal structure with a diameter of 54 nm, which was 10 times smaller than RxR in phosphatidylcholine liposomes. The small particle size of RxR-SMA allowed us to obtain scattering-less visible spectra with a high signal-to-noise ratio similar to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside. High-speed atomic force microscopy revealed that a single particle contained an average of 4.1 trimers of RxR (12.3 monomers). In addition, RxR-SMA showed a fast cyclic photoreaction (k = 13 s−1) comparable with RxR in phosphatidylcholine liposomes (17 s−1) but not to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside (0.59 s−1). By taking advantage of SMA, we determined the dissociation constant (Kd) of chloride for HsSRI as 34 mM. From these results, we conclude that SMA is a useful molecule forming a membrane-mimicking assembly for microbial rhodopsins having the advantages of detergents and liposomes.
AB - The membrane-embedded protein rhodopsin is widely produced in organisms as a photoreceptor showing a variety of light-dependent biological functions. To investigate its molecular features, rhodopsin is often extracted from cellular membrane lipids by a suitable detergent as “micelles.” The extracted protein is purified by column chromatography and then is often reconstituted into “liposomes” by removal of the detergent. The styrene-maleic acid (“SMA”) copolymer spontaneously forms nanostructures containing lipids without detergent. In this study, we applied SMA to characterize two microbial rhodopsins, a thermally stable rhodopsin, Rubrobacter xylanophilus rhodopsin (RxR), and an unstable one, Halobacterium salinarum sensory rhodopsin I (HsSRI), and evaluated their physicochemical properties in SMA lipid particles compared with rhodopsins in micelles and in liposomes. Those two rhodopsins were produced in Escherichia coli cells and were successfully extracted from the membrane by the addition of SMA (5 w/v %) without losing their visible color. Analysis by dynamic light scattering revealed that RxR in SMA lipid particles (RxR-SMA) formed a discoidal structure with a diameter of 54 nm, which was 10 times smaller than RxR in phosphatidylcholine liposomes. The small particle size of RxR-SMA allowed us to obtain scattering-less visible spectra with a high signal-to-noise ratio similar to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside. High-speed atomic force microscopy revealed that a single particle contained an average of 4.1 trimers of RxR (12.3 monomers). In addition, RxR-SMA showed a fast cyclic photoreaction (k = 13 s−1) comparable with RxR in phosphatidylcholine liposomes (17 s−1) but not to RxR in detergent micelles composed of n-dodecyl-β-D-maltoside (0.59 s−1). By taking advantage of SMA, we determined the dissociation constant (Kd) of chloride for HsSRI as 34 mM. From these results, we conclude that SMA is a useful molecule forming a membrane-mimicking assembly for microbial rhodopsins having the advantages of detergents and liposomes.
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U2 - 10.1016/j.bpj.2020.09.026
DO - 10.1016/j.bpj.2020.09.026
M3 - Article
C2 - 33086044
AN - SCOPUS:85093120126
SN - 0006-3495
VL - 119
SP - 1760
EP - 1770
JO - Biophysical Journal
JF - Biophysical Journal
IS - 9
ER -