Two-dimensional protein crystals on a solid substrate: effect of surface ligand concentration.
Proteins imbedded in solid-supported lipid bilayers can serve as model systems for investigations of cellular membranes and protein behavior on surfaces. We have investigated the self-assembly of streptavidin on mica-supported bilayer membranes. Using fluorescence microscopy and atomic force microscopy, our studies reveal that the concentration of surface ligand influences the molecular packing of the resulting protein arrays, which in turn affects overall crystal morphology. Two-dimensional streptavidin crystals are obtained when the biotinylated lipid density on the substrate reaches 1.5% mole fraction, yielding high-aspect morphologies that comprise primarily of crystals with P1 symmetry. At 3% and above, crystals with C222 symmetry are formed and result in H-shaped and confluent structures. In intermediate densities between 2 and 3%, a coexistence of P1 and C222 crystal forms is observed. The relationship between macroscopic morphology and molecular configuration is similar to previously reported data obtained at the air/water interface. This suggests that, under our experimental conditions, protein interactions with the supporting substrate are less significant for defining self-assembly behavior than interactions between protein molecules. Ligand-inhibition and fluorescence recovery after photobleaching were used to elucidate the concentration-dependent mechanism for the divergent crystal forms. We have measured the diffusion coefficient of molecules in P1-forming conditions to be approximately twice that of molecules in C222-forming concentrations, which is consistent with proteins bound to the surface through one and two ligands, respectively. The differential flexibility associated with the binding state is therefore likely to alter the subtle protein interactions involved in crystallization.
Lou
Chengfei
C
Department of Chemical Engineering and Materials Science, Developmental Biology Center, University of California, Irvine, California 92697, USA.
Wang
Zifu
Z
Wang
Szu-Wen
SW
D000538:Aluminum Silicates / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D003460:Crystallization; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties
D000538:Aluminum Silicates; D008024:Ligands; D008051:Lipid Bilayers; D011506:Proteins; D001710:Biotin; D019809:Streptavidin; C011934:mica