Rath Pragyan Parimita, Anand Gaurav, Agarwal Shalini
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India.
International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Bio Protoc. 2020 Feb 20;10(4):e3519. doi: 10.21769/BioProtoc.3519.
Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), Surface Plasmon Resonance (SPR) and many more. Among these, SPR stands out as a quick, label-free, reliable, and accurate quantitation technique. We have used SPR to elucidate the linkage between 14-3-3 Protein 3 (EhP3) and the actin cytoskeleton in the protist pathogen . It allowed us to screen EhP3 binding with several actin-binding/actin regulatory proteins (Coactosin, Actophorin, Twinfilin, Profilin, and Filamin). Our screening results suggested Coactosin as an important interacting partner of EhP3. A complete kinetic analysis indeed confirmed that EhCoactosin binds EhP3 with an affinity constant of 3 μM.
已知直接的蛋白质-蛋白质相互作用可调节广泛的细胞活动。为了解这些相互作用，人们可以采用各种实验方法，如动态光散射（DLS）、荧光共振能量转移（FRET）、等温滴定量热法（ITC）、化学交联、免疫共沉淀（Co-IP）、表面等离子体共振（SPR）等等。其中，SPR作为一种快速、无标记、可靠且准确的定量技术脱颖而出。我们利用SPR阐明了原生动物病原体中14-3-3蛋白3（EhP3）与肌动蛋白细胞骨架之间的联系。它使我们能够筛选EhP3与几种肌动蛋白结合/肌动蛋白调节蛋白（肌动蛋白结合蛋白、肌动蛋白运载蛋白、双丝肌动蛋白、肌动蛋白单体结合蛋白和细丝蛋白）的结合情况。我们的筛选结果表明肌动蛋白结合蛋白是EhP3的重要相互作用伙伴。完整的动力学分析确实证实，Eh肌动蛋白结合蛋白以3 μM的亲和常数结合EhP3。
