Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/18570
Título : Direct and noninvasive observation of two-dimensional nucleation behavior of protein crystals by advanced optical microscopy
Autor : Van Driessche, Alexander E. S., Sazaki, Gen, Otalora, Fermín, González-Rrico, Francisco M, Dold, Peter, Tsukamoto, Katsuo, Nakajima, Kazuo
Fecha de publicación : 2007
Editor: American Chemical Society
Resumen: We observed two-dimensional (2D) nucleation behavior on {110} and {101} faces of tetragonal crystals of model protein lysozyme by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). We measured, for the first time directly and noninvasively, the 2D nucleation rates using 99.99% pure lysozyme, 98.5% pure lysozyme (Seikagaku Co.), and 99.99% pure lysozyme with intentionally added impure proteins (fluorescent-labeled lysozyme, covalently bonded dimer of lysozyme, and 18 kDa polypeptide). We found that 2D nucleation was the dominant growth mechanism under conditions adopted in this study, and the 2D nucleation occurred randomly on the entire crystal surface irrespective of supersaturation within the range of a = In(C/Ce) = 0-1.4, where C is a bulk lysozyme concentration and Ce the solubility (crystal size: 0.2-0.3 mm). Repeated 2D nucleation, which continued for 3-4 layers, was also observed mainly when the impure proteins were present. In addition, multilayered 2D islands were formed after the adsorption of relatively large foreign particles on the crystal surface. From the comparison between the 2D nucleation rates determined on the {110} faces with and without the impure proteins, we concluded that homogeneous 2D nucleation occurred under a higher supersaturation range (a > 0.8), irrespective of the presence of the impurities. In contrast, under a lower supersaturation range (σ < 0.8), we found that significant heterogeneous 2D nucleation dominated the growth mainly when the impure proteins were present. The {101} faces exhibited more significant heterogeneous 2D nucleation induced by smaller amounts of impurities than in the case of the {110} faces. We also determined the ledge free energies of the homogeneous and heterogeneous nucleation. Within the experimental conditions used in this study, we could not find significant dependence of the ledge free energies of the heterogeneous nucleation on the kinds of impure proteins.
URI : http://hdl.handle.net/10261/18570
ISSN: 1528-7483
Citación : Crystal growth & design 7(10): 1528-7483 (2007)
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