Cao Yi, Balamurali M M, Sharma Deepak, Li Hongbin
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1.
Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15677-81. doi: 10.1073/pnas.0705367104. Epub 2007 Sep 25.
Protein-ligand interactions, including protein-protein interactions, are ubiquitously essential in biological processes and also have important applications in biotechnology. A wide range of methodologies have been developed for quantitative analysis of protein-ligand interactions. However, most of them do not report direct functional/structural consequence of ligand binding. Instead they only detect the change of physical properties, such as fluorescence and refractive index, because of the colocalization of protein and ligand, and are susceptible to false positives. Thus, important information about the functional state of protein-ligand complexes cannot be obtained directly. Here we report a functional single-molecule binding assay that uses force spectroscopy to directly probe the functional consequence of ligand binding and report the functional state of protein-ligand complexes. As a proof of principle, we used protein G and the Fc fragment of IgG as a model system in this study. Binding of Fc to protein G does not induce major structural changes in protein G but results in significant enhancement of its mechanical stability. Using mechanical stability of protein G as an intrinsic functional reporter, we directly distinguished and quantified Fc-bound and Fc-free forms of protein G on a single-molecule basis and accurately determined their dissociation constant. This single-molecule functional binding assay is label-free, nearly background-free, and can detect functional heterogeneity, if any, among protein-ligand interactions. This methodology opens up avenues for studying protein-ligand interactions in a functional context, and we anticipate that it will find broad application in diverse protein-ligand systems.
蛋白质-配体相互作用，包括蛋白质-蛋白质相互作用，在生物过程中普遍存在且至关重要，在生物技术中也有重要应用。已经开发出多种方法用于蛋白质-配体相互作用的定量分析。然而，其中大多数方法并未报告配体结合的直接功能/结构后果。相反，由于蛋白质和配体的共定位，它们仅检测物理性质的变化，如荧光和折射率，并且容易出现假阳性。因此，无法直接获得有关蛋白质-配体复合物功能状态的重要信息。在此，我们报告一种功能性单分子结合测定法，该方法使用力谱直接探测配体结合的功能后果并报告蛋白质-配体复合物的功能状态。作为原理验证，我们在本研究中使用蛋白G和IgG的Fc片段作为模型系统。Fc与蛋白G的结合不会在蛋白G中诱导主要的结构变化，但会导致其机械稳定性显著增强。利用蛋白G的机械稳定性作为内在功能报告分子，我们在单分子基础上直接区分并定量了Fc结合型和Fc游离型的蛋白G，并准确测定了它们的解离常数。这种单分子功能性结合测定法无需标记，几乎无背景，并且可以检测蛋白质-配体相互作用之间的功能异质性（如果存在的话）。这种方法为在功能背景下研究蛋白质-配体相互作用开辟了途径，我们预计它将在各种蛋白质-配体系统中得到广泛应用。
