Biomedical Research Centre, Sheffield Hallam University, Sheffield, United Kingdom.
Biophys J. 2011 Jul 20;101(2):504-11. doi: 10.1016/j.bpj.2011.06.011.
This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.
这项工作描述了一种新型质体伴侣受体 OEP61 与伴侣类型 Hsp70 和 Hsp90 的同工型之间的详细定量相互作用研究，使用全内反射椭圆光度法（TIRE）的光学方法。受体 OEP61 通过聚阳离子中间层静电固定在金表面上。TIRE 测量允许评估由于伴侣与受体蛋白结合而导致的吸附分子层的厚度变化。结果表明，只有 Hsp70 伴侣同工型而不是 Hsp90 能够与 OEP61 结合。进行了动态 TIRE 测量以评估上述反应的亲和常数，并且能够清楚地区分伴侣的特异性和非特异性结合以及高度相似的 Hsp70 同工型之间的结合特性差异。
