Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: Silane-polyethyleneglycol-lipid as a cushion and covalent linker

被引:458
作者
Wagner, ML [1 ]
Tamm, LK [1 ]
机构
[1] Univ Virginia, Dept Mol Physiol & Biol Phys, Charlottesville, VA 22908 USA
关键词
D O I
10.1016/S0006-3495(00)76392-2
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
There is increasing interest in supported membranes as models of biological membranes and as a physiological matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiological interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphology of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coefficients (0.8-1.2 x 10(-8) cm(2)/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b(5) and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were observed in the polymer-supported bilayers. Approximately 25% of cytochrome b(5) diffused with a diffusion coefficient of similar to 1 x 10(-8) cm(2)/s, and 50-60% diffused with a diffusion coefficient of similar to 2 x 10(-10) cm(2)/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of similar to 3 x 10(-9) cm(2)/s, and two-thirds diffused with a diffusion coefficient of similar to 4 x 10(-10) cm(2)/s. A model for the interaction of these proteins with the underlying polymer is discussed.
引用
收藏
页码:1400 / 1414
页数:15
相关论文
共 71 条
[61]   SURFACE STUDIES ON MULTICOMPONENT POLYMER SYSTEMS BY X-RAY PHOTOELECTRON-SPECTROSCOPY - POLYSTYRENE - POLY(ETHYLENE OXIDE) DIBLOCK COPOLYMERS [J].
THOMAS, HR ;
OMALLEY, JJ .
MACROMOLECULES, 1979, 12 (02) :323-329
[62]   Equilibrium, kinetics, diffusion and self-association of proteins at membrane surfaces: Measurement by total internal reflection fluorescence microscopy [J].
Thompson, NL ;
Drake, AW ;
Chen, LX ;
VandenBroek, W .
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 1997, 65 (01) :39-46
[63]   TOTAL INTERNAL-REFLECTION FLUORESCENCE MICROSCOPY - APPLICATION TO SUBSTRATE-SUPPORTED PLANAR MEMBRANES [J].
THOMPSON, NL ;
PEARCE, KH ;
HSIEH, HV .
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS, 1993, 22 (05) :367-378
[64]   Brownian ratchets: Molecular separations in lipid bilayers supported on patterned arrays [J].
van Oudenaarden, A ;
Boxer, SG .
SCIENCE, 1999, 285 (5430) :1046-1048
[65]   LATERAL DIFFUSION OF LIPIDS AND PROTEINS IN BILAYER-MEMBRANES [J].
VAZ, WLC ;
GOODSAIDZALDUONDO, F ;
JACOBSON, K .
FEBS LETTERS, 1984, 174 (02) :199-207
[66]   THE CARBOXYL-TERMINUS OF THE MEMBRANE-BINDING DOMAIN OF CYTOCHROME B(5) SPANS THE BILAYER OF THE ENDOPLASMIC-RETICULUM [J].
VERGERES, G ;
RAMSDEN, J ;
WASKELL, L .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (07) :3414-3422
[67]   3-DIMENSIONAL STRUCTURE OF MEMBRANE-BOUND ANNEXIN-V - A CORRELATIVE ELECTRON MICROSCOPY-X-RAY CRYSTALLOGRAPHY STUDY [J].
VOGES, D ;
BERENDES, R ;
BURGER, A ;
DEMANGE, P ;
BAUMEISTER, W ;
HUBER, R .
JOURNAL OF MOLECULAR BIOLOGY, 1994, 238 (02) :199-213
[68]   SUPPORTED PHOSPHOLIPID-BILAYERS PREPARED BY THE LB/VESICLE METHOD - A FOURIER-TRANSFORM INFRARED ATTENUATED TOTAL-REFLECTION SPECTROSCOPIC STUDY ON STRUCTURE AND STABILITY [J].
WENZL, P ;
FRINGELI, M ;
GOETTE, J ;
FRINGELI, UP .
LANGMUIR, 1994, 10 (11) :4253-4264
[69]   Polymer-cushioned bilayers. I. A structural study of various preparation methods using neutron reflectometry [J].
Wong, JY ;
Majewski, J ;
Seitz, M ;
Park, CK ;
Israelachvili, JN ;
Smith, GS .
BIOPHYSICAL JOURNAL, 1999, 77 (03) :1445-1457
[70]   Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus [J].
Wong, JY ;
Park, CK ;
Seitz, M ;
Israelachvili, J .
BIOPHYSICAL JOURNAL, 1999, 77 (03) :1458-1468