Department of Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University, VU Station B 351822, Nashville, Tennessee 37235-1822, USA.
Anal Chem. 2009 Mar 1;81(5):1865-71. doi: 10.1021/ac802327h.
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 BSI 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 betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-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 BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.
我们使用背向散射干涉法（BSI）在皮升级别、无标记的溶液中报告了蛋白质-蛋白质相互作用的定量分析。在 PDMS 微芯片上引入溶液后，测量折射率的变化，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变 BSI 轨迹，以表征小分子热休克蛋白 alpha-Crystallin 与两种底物的相互作用：T4 溶菌酶的去稳定突变体和体内靶标 betaB1-Crystallin。结果再现了 alphaB-Crystallin 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 alphaA-Crystallin 突变体与 betaB1-Crystallin 的结合被激活。从 BSI 信号的稳态值获得的结合等温线得出了有意义的解离常数，并确立 BSI 作为一种新工具，用于在生理条件下使用极小的样品量快速识别分子伴侣。这项工作表明，BSI 可以扩展到筛选与疾病相关的突变体文库，以定量测量结合亲和力和/或动力学的变化。
