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Langmuir 2003, 19, 842-850
Formation of Supported Lipid Bilayer Membranes on SiO2 from Proteoliposomes Containing Transmembrane Proteins Annette Grane´li,*,†,‡ Jan Rydstro¨m,‡ Bengt Kasemo,† and Fredrik Ho¨o¨k*,† Department of Chemical Physics, Applied Physics, Chalmers, Fysikgra¨ nd 3, S-41296 Go¨ teborg, Sweden, and Department of Biochemistry and Biophysics, Go¨ teborg University, Go¨ teborg, Sweden Received July 12, 2002. In Final Form: October 23, 2002 We report the preparation of protein-containing supported phospholipid bilayers (SPBs) on silica (SiO2). The bilayers are formed from small proteoliposomes, which convert to an SPB when the liposomes adsorb on the surface. The kinetics of this conversion process was followed in real time, using the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. The proteoliposomes were prepared by reconstitution of two different proteins into small unilamellar liposomes (diameter ∼ 26 nm), creating proteoliposomes with diameters ranging from ca. 50 to 85 nm, depending on protein concentration. The two proteins were proton translocating nicotinamide nucleotide transhydrogenase (TH) from Escherichia coli and gramicidin A (GrA) from Bacillus brevis. The SPB formation process was measured and compared for different protein situations in the liposomes: (i) with the intact TH in the proteoliposomes, (ii) after removal of the water-exposed, hydrophilic domains of TH, and (iii) with GrA-containing proteoliposomes (with no water-soluble domains). In the latter two cases qualitatively similar kinetics were observed as with pure (i.e., without proteins) liposomes. In contrast, the waterexposed hydrophilic domains on TH are found to partially hamper the SPB formation process leaving fractions of nonruptured proteoliposomes on the surface. The latter effect becomes stronger with increasing protein/lipid ratio in the proteoliposomes. A comparison was made between activity measurements of TH-containing proteoliposomes in solution and TH-containing SPBs. The latter results support the conclusions from the QCM-D and SPR measurements.
Introduction Supported phospholipid bilayers (SPBs), functionalized with membrane constituents, are interesting cell-mimicking templates for studies of, e.g., membrane-mediated processes,1 cell-cell communication,2 bioelectronics,3,4 development of drug-screening,5 and biosensor devices.6 In addition, SPBs aid in the scientific task of characterizing the physical and chemical properties of natural cell membranes and in fundamental studies of the structure and function of membrane proteins.3-4,7-10 It is well established that exposure of SiO2, glass, and mica surfaces to phospholiposome suspensions leads to spontaneous formation of planar, extended, stable (if fully * Corresponding authors. Telephone: +46-31-7723457. Fax: +46-31-7723134. E-mail:
[email protected] (A.G.). Telephone: +46-31-7723464. Fax: +46-31-7723134. E-mail:
[email protected] (F.E.). † Chalmers. ‡ Go ¨ teborg University. (1) Sackmann, E. Science 1996, 271, 43-48. (2) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys. Acta 1986, 864, 95-106. (3) Lindholm-Sethson, B.; Carrasco Gonzalez, J.; Puu, G. Langmuir 1998, 14, 6705-6708. (4) Burgess, J. D.; Rhoten, M. C.; Hawkridge, F. M. J. Am. Chem. Soc. 1998, 120, 4488-4491. (5) Schmid, E. L.; Tairi, A. P.; Hovius, R.; Vogel, H. Anal. Chem. 1998, 70, 1331-1338. (6) Gizeli, E.; Liley, M.; Lowe, C. R.; Vogel, H. Anal. Chem. 1997, 69, 4808-4813. (7) Gritsch, S.; Nollert, P.; Jahnig, F.; Sackmann, E. Langmuir 1998, 14, 3118-3125. (8) Heyse, S.; Ernst, O. P.; Dienes, Z.; Hofmann, K. P.; Vogel, H. Biochemistry 1998, 37, 507-522. (9) Liley, M.; Bouvier, J.; Vogel, H. J. Colloid Interface Sci. 1997, 194, 53-58. (10) Kro¨ger, D.; Hucho, F.; Vogel, H. Anal. Chem. 1999, 71, 31573165.
hydrated), and fluid lipid bilayers. This has been studied by quartz crystal microbalance (QCM-D),11,12 surface plasmon resonance (SPR),11 atomic force microscopy (AFM),13,14 and fluorescence recovery after photobleaching (FRAP)15 measurements. The detailed kinetics of the formation process is sensitive to temperature,16 liposome size,13,17 Ca2+ concentration,18 lipid composition,18 and insertion of a low number (
