TY - JOUR
T1 - A concise method for quantitative analysis of interactions between lipids and membrane proteins
AU - Inada, Masataka
AU - Kinoshita, Masanao
AU - Sumino, Ayumi
AU - Oiki, Shigetoshi
AU - Matsumori, Nobuaki
N1 - Funding Information:
We thank Professor Michio Murata (Osaka University) for his help in halobacterium culture, and Associate Professor Ayami Matsushima (Kyushu University) for her help in SPR measurements. M. Kinoshita acknowledges JSPS KAKENHI ( JP17K15107 ) for funding. A. Sumino acknowledges JST PRESTO, the Shiseido Female Researcher Science Grant, JSPS KAKENHI ( JP17H05058 and JP16K15178 ) for funding. S. Oiki acknowledges JSPS KAKENHI ( JP17H04017 and JP16K15179 ) and MEXT KAKENHI ( JP16H00759 ). N. Matsumori acknowledges JSPS KAKENHI ( JP15H03121 ), MEXT KAKENHI ( JP16H00773 ), and JST ERATO (Lipid Active Structure Project).
Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/6/20
Y1 - 2019/6/20
N2 - Although interactions between lipids and membrane proteins (MPs) have been considered crucially important for understanding the functions of lipids, lack of useful and convincing experimental methods has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance (SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip surface with self-assembled monolayer (SAM) with C 6 -chain. To develop this method, we used bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits of using C 6 -SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM provides immobilized MPs with a partial membranous environment, which is important for the stabilization of MPs; and (3) a thinner C 6 -SAM layer (1 nm) compared with MP size forces the MP to bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C 6 -SAM is 10 times higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation, demonstrating the validity and utility of this method.
AB - Although interactions between lipids and membrane proteins (MPs) have been considered crucially important for understanding the functions of lipids, lack of useful and convincing experimental methods has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance (SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip surface with self-assembled monolayer (SAM) with C 6 -chain. To develop this method, we used bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits of using C 6 -SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM provides immobilized MPs with a partial membranous environment, which is important for the stabilization of MPs; and (3) a thinner C 6 -SAM layer (1 nm) compared with MP size forces the MP to bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C 6 -SAM is 10 times higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation, demonstrating the validity and utility of this method.
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U2 - 10.1016/j.aca.2019.01.042
DO - 10.1016/j.aca.2019.01.042
M3 - Article
C2 - 30876624
AN - SCOPUS:85061588629
VL - 1059
SP - 103
EP - 112
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
SN - 0003-2670
ER -