Free-Solution Label-Free Detection of α-Crystallin Chaperone Interactions by Back-Scattering Interferometry
Free-Solution Label-Free Detection of α-Crystallin Chaperone Interactions by Back-Scattering Interferometry
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DOI：
10.1021/ac802327h
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发表时间：
2009-03-01
影响因子：
7.4
通讯作者：
Mchaourab, Hassane S.
中科院分区：
文献类型：
--
作者：
Latham, Joey C.;Stein, Richard A.;Mchaourab, Hassane S.
我们报告了使用反向散射干涉测量法（BSI）对皮升体积内游离溶液中蛋白质-蛋白质相互作用的定量、无标记分析。测量引入 PDMS 微芯片上的溶液的折射率变化，从而确定双模式结合的正向和反向速率常数。使用全局分析方法直接拟合时间依赖性 BST 迹线，以表征小热休克蛋白 α-晶状体蛋白与两种底物的相互作用：T4 溶菌酶的不稳定突变体和体内目标 β B1-晶状体蛋白。结果概括了 α B-晶状体蛋白根据其解折叠自由能差异结合 T4L 突变体的选择性。此外，我们证明与遗传性白内障相关的 α A-晶状体蛋白突变体已激活与 β B1-晶状体蛋白的结合。从 BSI 信号的稳态值获得的结合等温线产生了有意义的解离常数，并将 BSI 确立为一种在生理条件下使用极少量样品快速识别分子伴侣的新工具。这项工作表明，BST 可以扩展到筛选疾病相关突变体文库，以量化亲和力和/或结合动力学的变化。
We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BST traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target beta B1-Crystallin. The results recapitulate the selectivity of alpha B-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alpha A-Crystallin mutant linked to hereditary cataract has activated binding to beta B1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BST can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.