ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(4): 391-395 CN 31-1300/Q Preparation of Liposome Containing Bacteriorhodopsin with Natural Preferred Orientation of Its Transient Photoresponse HUANG Li, MING Ming, LIU Jia, LIU Jian, LI Qing-Guo 1, DING Jian-Dong* ( Department of Macromolecular Science, Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China; 1 Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China ) Abstract Bacteriorhodopsin is a membrane protein of halobacteria and functions as a light-driven protein pump. After we isolated br from cultured halobacteria, br was mixed with amphiphilic DPPC under different ph. The liposomes were formed after sonication. The remaining biological activity of br as a proton pump was then verified and pulsed-light-induced proton movement was detected, while liposomes were observed via TEM and light scattering. Although there was no noticeable difference in morphologies of both vesicles formed at ph=2.5 and ph=7.0, the orientations of br in both liposomes were found to be opposite under these two conditions. This experiment confirmed that the protein br, when self-assembling into liposomes under acid medium, kept the similar orientation as in the natural plasmid membrane. Such a normal orientation was, however, different from most of reports in the literature about liposomes prepared under normal neutral conditions. Key words bacteriorhodopsin; membrane protein; self assembly; liposome Received: December 9, 2002 Accepted: January 27, 2003 This work was supported by grants from the National Natural Science Foundation of China(No. 29825109, No. 20174006), the Award Foundation for Young Teachers from Ministry of Education, the Major State Basic Research Development Program of China (973 Program)(No. G1999054306-03), the National High Technology Research and Development Program of China(863 Program) (No. 2001AA215451), and Science & Technology Developing Foundation of Shanghai (No. 02DZ11010) *Corresponding author: Tel, 86-21-65643506, 65642531; Fax, 86-21-65640293; e-mail, jdding1@fudan.edu.cn 1 * (, 200433; 1, 200433 )
(br),,,, br, ph=2.5 br DPPC br, br, br DPPC ; br,, ; ; ; (bacteriorhodopsin, br) (Halobacterium salinarium),, 1 br,, (alltrans) 13- (13-cis),, (, 412 nm M ),, [1,2],, pka, N, C,, [3], br N, C [1 4] Fig.1 The seven trans-membrane a-helix structure of br molecule 60,,, 2,, 5 7 nm, 25 500 nm
Fig.2 Schematic presentation of liposome containing membrane protein with different preferred orientation, br, br [4,5], C, N ; [6] br br, br (EPC)[7], [8] br, br 1 (Material and Methods) 1.1 br R1M1 [7,9]; ph pyranine(8-hydroxy- 1,3,6-pyrenetrisulfonate) Kadak ; (dipalmitoylphosphatidylcholine, DPPC) Sigma 1.2 br DPPC [7], (2:1) DPPC ( 10 g/l) 1 ml 50, N2,, br 3 ml( 1:15 ) 15 ml 0.1 mol/l NaCl,, 15 s, 30 s, 4 3 br 1 mol/l HCl ph=2.5,, 15 min,, 15 s, 30 s, 4 3 2 (,, 200 W),, br 69 000 g 1 h,, br ; 0.1 mol/l NaCl 1 mol/l NaOH ph, 31 000 g 25 min, ( br ), br DPPC 1.3 UV-260 M, [1 ], 1 ms,, 250 W,, *.wsv ASCII
, Origin 2 (Results and Discussion) 2.1, 5 min, ( ), ( 20 000 150 000 ), 3; (,, ) ( ), Fig.3 Observation of DPPC liposomes containing br (A) TEM and (B) dynamic laser scattering. The fed molar ratio of DPPC to br was 15 :1. 2.2 br br (, 568 nm ; ) 412 nm, M, 4, br, br, ( 3 )
Fig.4 The decay curve of photo -intermediate M412 of DPPC -liposomes containing br formed at ph=2.5 and detected at neutral ph 2.3 ph br, br [11,12], br br, ph ph pyranine br ph 458 nm;, M,,,, A458 ph ph 55(A) 2, 1, 2 1 5(B) 2, ph A458, brn, br N, C, Fig.5 Flash -induced absorption change of DPPC -liposomes containing br formed at ph=2.5 and detected at neutral ph (A) Absorption response before (curve 1) and after (curve 2) adding the ph sensitive dye pyranine; (B) the resultant difference absorption curve (curve 2) and that from the control experiment (curve 1), which corresponds to DPPC liposomes without br. (ph=7.0) br 6,,, br C, (DPPC,, )
Fig.6 Flash -induced absorption change of EPC-liposomes containing br formed at ph=7.0 (A) Absorption response before (curve 1) and after (curve 2) adding the ph sensitive dye pyranine; (B) the resultant difference absorption curve. 5 6,, ;,, [1 ] ;, ph, [13] br,? br ph, br References 1 Grigorieff N, Ceska TA, Downing KH, Baldwin JM, Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. [WT X J Mol Biol, 1996, 259: 393 421 2 Zimanyi L, Cao Y, Needleman R, Ottolenghi M, Lanyi JK. Pathway of proton uptake in the bacteriorhodopsin photocycle. [WT X Biochemistry, 1993, 32: 7669 7678 3 Brown LS, Dioumaev AK, Needleman R, Lanyi JK. Local-access model for proton transfer in bacteriorhodopsin. [WT X ]Biochemistry, 1998, 37: 3982 3993 4 Gerber GE, Gray CP, Wildenauer D, Khorana HG. Orientation of bacteriorhodopsin in Halobacterium halobium as studied by selective proteolysis. [WT X Proc Natl Acad Sci USA, 1977, 74(12): 5426 5430 5 Monika H, Ron M, Peter O. Direction of proton translocation in proteoliposomes formed from purple membrane and acidic lipids depend on the ph during reconstitution. [WT X ]Biochim Biophys Acta, 1977, 465: 415 420 6 Niu L, Kim JM, Khorana HG. Structure and function in rhodopsin: Asymmetric reconstitution of rhodopsin in liposomes, Proc Natl Acad Sci USA, 2002, 99:13409-13412 7 Liu J, Zhu Y, Li QG, Ding JD. Preparation of phospholipids vesicles containing purple membrane and detection of the proton pumping function. J Fudan Univ, 1999, 38(5): 549-552 8 Liu WL, Zhang ZH eds. Membrane and Liposome Technology, Changsha: Hunan Technology Publishers, 1986(in Chinese) 9 Becher B, Cassim JY. Improved isolation procedures for the purple membrane of, 2001, 33(4): 443 446 10 Liu J, Ming M, Liu J, Huang L, Li QG, Ding JD. Preparation and study of bacteriohodopsin / poly (vinyl
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