Biophys J, September 2000, p. 1400-1414, Vol. 79, No. 3

Tethered Polymer-Supported Planar Lipid Bilayers for Reconstitution of Integral Membrane Proteins: Silane-Polyethyleneglycol-Lipid as a Cushion and Covalent Linker

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908-0736 USA

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 × 10⁸ cm²/s) were observed when the polymer concentration was kept slightly below the mushroom-to-brush transition. Cytochrome b₅ 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 b₅ diffused with a diffusion coefficient of ~ 1 × 10⁸ cm²/s, and 50-60% diffused with a diffusion coefficient of ~2 × 10¹⁰ cm²/s. Similarly, one-third of annexin V diffused with a diffusion coefficient of ~ 3 × 10⁹ cm²/s, and two-thirds diffused with a diffusion coefficient of ~4 × 10¹⁰ cm²/s. A model for the interaction of these proteins with the underlying polymer is discussed.

Biophys J, September 2000, p. 1400-1414, Vol. 79, No. 3
© 2000 by the Biophysical Society   0006-3495/00/09/1400/15  $2.00

This article has been cited by other articles:

				T. C. Anglin and J. C. Conboy Lateral Pressure Dependence of the Phospholipid Transmembrane Diffusion Rate in Planar-Supported Lipid Bilayers Biophys. J., July 1, 2008; 95(1): 186 - 193. [Abstract] [Full Text] [PDF]

				V. Kiessling, J. M. Crane, and L. K. Tamm Transbilayer Effects of Raft-Like Lipid Domains in Asymmetric Planar Bilayers Measured by Single Molecule Tracking Biophys. J., November 1, 2006; 91(9): 3313 - 3326. [Abstract] [Full Text] [PDF]

				T. Cha, A. Guo, and X.-Y. Zhu Formation of Supported Phospholipid Bilayers on Molecular Surfaces: Role of Surface Charge Density and Electrostatic Interaction Biophys. J., February 15, 2006; 90(4): 1270 - 1274. [Abstract] [Full Text] [PDF]

				K. Tawa and K. Morigaki Substrate-Supported Phospholipid Membranes Studied by Surface Plasmon Resonance and Surface Plasmon Fluorescence Spectroscopy Biophys. J., October 1, 2005; 89(4): 2750 - 2758. [Abstract] [Full Text] [PDF]

				J. Liu and J. C. Conboy 1,2-Diacyl-Phosphatidylcholine Flip-Flop Measured Directly by Sum-Frequency Vibrational Spectroscopy Biophys. J., October 1, 2005; 89(4): 2522 - 2532. [Abstract] [Full Text] [PDF]

				M. A. Deverall, E. Gindl, E.-K. Sinner, H. Besir, J. Ruehe, M. J. Saxton, and C. A. Naumann Membrane Lateral Mobility Obstructed by Polymer-Tethered Lipids Studied at the Single Molecule Level Biophys. J., March 1, 2005; 88(3): 1875 - 1886. [Abstract] [Full Text] [PDF]

				K. Bacia, C. G. Schuette, N. Kahya, R. Jahn, and P. Schwille SNAREs Prefer Liquid-disordered over "Raft" (Liquid-ordered) Domains When Reconstituted into Giant Unilamellar Vesicles J. Biol. Chem., September 3, 2004; 279(36): 37951 - 37955. [Abstract] [Full Text] [PDF]

				T. Hughes, B. Strongin, F. P. Gao, V. Vijayvergiya, D. D. Busath, and R. C. Davis AFM Visualization of Mobile Influenza A M2 Molecules in Planar Bilayers Biophys. J., July 1, 2004; 87(1): 311 - 322. [Abstract] [Full Text] [PDF]

				C. C. Ng, Y.-L. Cheng, and P. S. Pennefather Properties of a Self-Assembled Phospholipid Membrane Supported on Lipobeads Biophys. J., July 1, 2004; 87(1): 323 - 331. [Abstract] [Full Text] [PDF]

				J. Tong and T. J. McIntosh Structure of Supported Bilayers Composed of Lipopolysaccharides and Bacterial Phospholipids: Raft Formation and Implications for Bacterial Resistance Biophys. J., June 1, 2004; 86(6): 3759 - 3771. [Abstract] [Full Text] [PDF]

				B. L. Stottrup, S. L. Veatch, and S. L. Keller Nonequilibrium Behavior in Supported Lipid Membranes Containing Cholesterol Biophys. J., May 1, 2004; 86(5): 2942 - 2950. [Abstract] [Full Text] [PDF]

				J. M. Crane and L. K. Tamm Role of Cholesterol in the Formation and Nature of Lipid Rafts in Planar and Spherical Model Membranes Biophys. J., May 1, 2004; 86(5): 2965 - 2979. [Abstract] [Full Text] [PDF]

				V. Kiessling and L. K. Tamm Measuring Distances in Supported Bilayers by Fluorescence Interference-Contrast Microscopy: Polymer Supports and SNARE Proteins Biophys. J., January 1, 2003; 84(1): 408 - 418. [Abstract] [Full Text] [PDF]