Department of Physics, Concordia University, Montreal Qc, Canada.
Department of Chemistry and Biochemistry, Concordia University, Montreal Qc, Canada.
PLoS One. 2019 Jan 17;14(1):e0206338. doi: 10.1371/journal.pone.0206338. eCollection 2019.
The native octameric structure of streptococcal enolase from Streptococcus pyogenes increasingly dissociates as amino acid residues are removed one by one from the carboxy-terminus. These truncations gradually convert native octameric enolase into monomers and oligomers. In this work, we investigated how these truncations influence the interaction between Streptococcal enolase and canine plasminogen. We used dual polarization interferometry (DPI), localized surface plasmon resonance (LSPR), and sedimentation velocity analytical ultracentrifugation (AUC) to study the interaction. The DPI was our first technique, was performed on all the truncations and used one exclusive kind of chip. The LSRP was used to show that the DPI results were not dependent on the type of chip used. The AUC was required to show that our surface results were not the result of selecting a minority population in any given sample; the majority of the protein was responsible for the binding phenomenon we observed. By comparing results from these techniques we identified one detail that is essential for streptococcal enolase to bind plasminogen: In our hands the individual monomers bind plasminogen; dimers, trimers, tetramers may or may not bind, the fully intact, native, octamer does not bind plasminogen. We also evaluated the contribution to the equilibrium constant made by surface binding as well as in solution. On a surface, the association coefficient is about twice that in solution. The difference is probably not significant. Finally, the fully octameric form of the protein that does not contain a hexa-his N-terminal peptide does not bind to a silicon oxynitride surface, does not bind to an Au-nanoparticle surface, does not bind to a surface coated with Ni-NTA nor does it bind to a surface coated with DPgn. The likelihood is great that the enolase species on the surface of Streptococcus pyogenes is an x-mer of the native octamer.
来自酿脓链球菌的烯醇化酶的天然八聚体结构随着从羧基末端逐个去除氨基酸残基而逐渐解离。这些截断逐渐将天然八聚体烯醇化酶转化为单体和低聚物。在这项工作中，我们研究了这些截断如何影响链球菌烯醇化酶与犬纤溶酶原之间的相互作用。我们使用双折射干涉（DPI）、局部表面等离子体共振（LSPR）和沉降速度分析超速离心（AUC）来研究相互作用。DPI 是我们的第一种技术，对所有截断都进行了操作，并使用了一种独特的芯片。LSRP 用于表明 DPI 结果不依赖于所用芯片的类型。AUC 用于表明我们的表面结果不是选择给定样品中少数群体的结果；大多数蛋白质负责我们观察到的结合现象。通过比较这些技术的结果，我们确定了链球菌烯醇化酶与纤溶酶原结合的一个细节是必不可少的：在我们手中，单个单体结合纤溶酶原；二聚体、三聚体、四聚体可能结合也可能不结合，完全完整的、天然的、八聚体不结合纤溶酶原。我们还评估了表面结合以及在溶液中对平衡常数的贡献。在表面上，结合系数约为溶液中的两倍。差异可能并不显著。最后，不含六组氨酸 N 端肽的完全八聚体形式的蛋白质既不与氮化硅表面结合，也不与 Au 纳米颗粒表面结合，既不与 Ni-NTA 涂层表面结合，也不与 DPgn 涂层表面结合。链球菌烯醇化酶在酿脓链球菌表面的存在形式很可能是天然八聚体的 x 聚体。
