Quantitative analysis of multivalent ligand presentation on gold glyconanoparticles and the impact on lectin binding.
Quantitative analysis of multivalent ligand presentation on gold glyconanoparticles and the impact on lectin binding.
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DOI：
10.1021/ac102114z
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发表时间：
2010-11-01
影响因子：
7.4
通讯作者：
Yan, Mingdi
中科院分区：
文献类型：
--
作者：
Wang, Xin;Ramstrom, Olof;Yan, Mingdi
糖金属材料是一种携带多个碳水化合物配体的纳米材料，为敏感蛋白质的识别提供了一个极好的平台。利用纳米材料作为支架，已经证明了多糖配体和蛋白质之间的多价相互作用。然而，缺乏对这些糖链物质结合亲和力的定量分析。在这篇文章中，我们报道了一种新的方法来测量糖核蛋白(GNP)与蛋白质相互作用的亲和力，该方法基于荧光竞争结合分析，得到了GNP与相互作用蛋白质的表观解离常数(Kd)。利用我们最近发展起来的光偶联化学方法，合成了与未衍生化的单糖、寡糖、多糖结合的金纳米颗粒。测定了这些GNPs与凝集素的亲和力，比相应的自由配体与凝集素的亲和力高几个数量级。进一步研究了配体显示对GNPs结合亲和力的影响，制备了不同连接基类型、间隔基长度、配体密度和纳米颗粒尺寸的GNPs，并测定了Kd值。含有碳氢化合物和环氧乙烷单元的长间隔基链具有最高的结合亲和力和检测灵敏度。结合亲和力一般随着配基密度的增加而增加，在低配基密度时亲和力急剧增加。此外，在较小的NP上亲和力的增强比在较大的NPs上更明显。这些结果不仅表明GNPs的结合亲和力受配体在纳米颗粒上的呈现方式的影响很大，而且还为通过配体展示的方式定制亲和力可调的糖金属材料铺平了道路。
Glyconanomaterials, nanomaterials carrying multiple carbohydrate ligands, provide an excellent platform for sensitive protein recognition. Using nanomaterials as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, the quantitative analysis of the binding affinity of these glyconanomaterials has been lacking. In this article, we report a new method to measure the binding affinity of glyconanoparticle (GNP)-protein interactions based on a fluorescent competition binding assay, which yielded the apparent dissociation constant (Kd) of GNPs with the interacting protein. Au nanoparticles conjugated with underivatized mono-, oligo-, poly-saccharides were synthesized using our recently developed photocoupling chemistry. The affinities of these GNPs with lectins were measured and were several orders of magnitude higher than the corresponding free ligands with lectins. The effect of ligand display on the binding affinity of GNPs was furthermore studied where GNPs of varying linker type, spacer length, ligand density, and nanoparticle size were prepared and Kd values determined. The long spacer linker containing hydrocarbon and ethylene oxide units gave the highest binding affinity as well as assay sensitivity. The binding affinity increased with ligand density in general, showing a drastic increase in affinity at low ligand density. In addition, the affinity enhancement was more pronounced on smaller NPs than the larger ones. These results not only demonstrate that the binding affinity of GNPs is highly influenced by how the ligands are presented on the nanoparticles, but also pave the way for tailor-made glyconanomaterials with tunable affinity by way of ligand display.
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