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首页> 外文期刊>Crystal growth & design >Direct and noninvasive observation of two-dimensional nucleation behavior of protein crystals by advanced optical microscopy
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Direct and noninvasive observation of two-dimensional nucleation behavior of protein crystals by advanced optical microscopy

机译：通过高级光学显微镜直接和无创地观察蛋白质晶体的二维成核行为

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摘要

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 IS 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 sigma = ln(C/C-e) = 0-1.4, where C is a bulk lysozyme concentration and C, 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 (sigma > 0.8), irrespective of the presence of the impurities. In contrast, under a lower supersaturation range (sigma < 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.
机译：我们通过激光共聚焦显微镜与微分干涉对比显微镜（LCM-DIM）相结合，观察了模型蛋白溶菌酶四方晶体的{110}和{101}面上的二维（2D）成核行为。我们首次直接和无创地测量了使用99.99％纯溶菌酶，98.5％纯溶菌酶（Seikagaku Co.）和99.99％纯溶菌酶以及有意添加的不纯蛋白质（荧光标记的溶菌酶，共价结合的二聚体）的二维成核率（溶菌酶和IS kDa多肽）。我们发现，在本研究采用的条件下，二维成核是主要的生长机制，并且二维成核在整个晶体表面上随机发生，而不管在sigma = ln（C / Ce）= 0-1.4范围内的过饱和状态，其中C是溶菌酶的总浓度，C是溶解度（晶体大小：0.2-0.3 mm）。主要在存在不纯蛋白的情况下也观察到持续3-4层的重复2D成核。另外，在晶体表面上吸附了较大的异物之后，形成了多层2D岛。通过比较在有和没有杂质蛋白质的情况下在{110}面上确定的2D成核率，我们得出结论，无论杂质的存在与否，均一的2D成核在较高的过饱和范围内发生（σ> 0.8）。相反，在较低的过饱和范围（sigma <0.8）下，我们发现，当存在不纯蛋白质时，显着的异质2D成核作用主导了生长。与{110}面相比，{101}面表现出由较少量的杂质引起的更显着的异质2D成核。我们还确定了均匀和不均匀成核的壁架自由能。在这项研究中使用的实验条件下，我们找不到异质成核的壁架自由能对不纯蛋白质种类的显着依赖性。

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