1794
Langmuir 2003, 19, 1794-1801
Effect of Ionic Strength on Two-Dimensional Streptavidin Crystallization† Pasut Ratanabanangkoon‡ and Alice P. Gast*,§ Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, and Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received July 1, 2002 We study two-dimensional (2D) crystallization of streptavidin under low ionic strength conditions. We investigate crystals on both giant unilamellar lipid bilayer vesicles and lipid monolayers at the air-water interface. In contrast to 2D crystallization in high ionic strength buffers, the electrostatic repulsions under these conditions favor the low density C222 crystal form. The crystals grown on giant vesicles and those on lipid monolayers show the same crystal structure and similar crystallization trends despite the difference in surface geometries. When the ionic strength of the crystallization solution is progressively increased at pH 4, we observe a transition from C222 to the denser P1 space group commonly seen in high ionic strength conditions at this pH. A new crystal structure appears at intermediate ionic strength, illustrating the role of electrostatic repulsions in these ordering processes.
I. Introduction Two-dimensional (2D) protein crystals on various lipid surfaces have been used as model systems to study the fundamentals of protein-protein interactions, protein selfassembly, and 2D phase transitions.1-7 A better understanding of such phenomena is strongly desired and can yield numerous benefits including better protein crystals for structural studies, the creation and manipulation of supported membranes for biosensors, and the means to make biomimetic protein coated structures. There have been numerous studies toward the development of methods to efficiently immobilize biomolecules onto inorganic surfaces, usually through supported bilayers.27 The protein streptavidin has been studied for such purposes due to its unusually high binding affinity to biotin.8,9 Streptavidin bound to biotin-functionalized lipid bilayers on solid surfaces serves as an efficient linker. With biotin-binding sites on opposite sides of the molecule, one side of the molecule can be bound to the bilayer surface, while the other is available to immobilize other biotinfunctionalized biomolecules. * To whom correspondence should be addressed. Mailing address: Alice P. Gast, 3-240, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 2531403. Fax: (617)-253-8388. E-mail,
[email protected]. † Part of the Langmuir special issue entitled The Biomolecular Interface. ‡ Stanford University. § Massachusetts Institute of Technology. (1) Darst, S. A.; Ahlers, M.; Meller, P. H.; Kubalek, E. W.; Blankenburg, R.; Ribi, H. O.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991, 59, 387-396. (2) Kornberg, R. D.; Darst, S. A. Curr. Opin. Struct. Biol. 1991, 1, 642-646. (3) Wang, S. W.; Robertson, C. R.; Gast, A. P. Langmuir 1999, 15, 1541-1548. (4) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989, 243, 85-88. (5) Wetzer, B.; Pfandler, A.; Gyo¨rvary, E.; Pum, D.; Lo¨sche, M.; Sleytr, U. B. Langmuir 1998, 14, 6899-6906. (6) Sa´ra, M.; Sleytr, U. B. J. Bacteriol. 2000, 182, 859-868. (7) Reviakine, I.; Bergsma-Schutter, W.; Morozov, A. N.; Brisson, A. Langmuir 2001, 17, 1680-1686. (8) Bayer, E. A.; Benhur, H.; Wilchek, M. Methods Enzymol. 1990, 184, 80-89. (9) Green, N. M. Methods Enzymol. 1990, 184, 51-67.
Crystallization of streptavidin on biotinylated lipid monolayers has been extensively studied in relatively high ionic strength (
