Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada.
State Key Laboratory of Precision Measurements Technology and Instruments, School of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin, 300072, China.
Angew Chem Int Ed Engl. 2017 May 22;56(22):6117-6121. doi: 10.1002/anie.201610648. Epub 2016 Dec 27.
Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2) as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2) lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.
单分子力谱（SMFS）已成为研究蛋白质在单分子水平上机械展开/折叠的有力工具。由串联相同重复序列组成的多蛋白已广泛应用于基于原子力显微镜（AFM）的 SMFS 研究中，其中多蛋白不仅可作为识别单分子拉伸事件的指纹，还可能提高数据收集的统计学意义。然而，此类实验的固有假设是多蛋白中的所有结构域都是等效的，且拉伸由 n 个结构域组成的多蛋白的一个 SMFS 轨迹等同于拉伸单个结构域的 n 个轨迹。这种假设尚未通过实验验证。本研究以小蛋白 NuG2 及其多蛋白（NuG2）为模型系统，使用光阱（OT）技术直接验证了这一假设。结果表明，OT 实验对 NuG2 和（NuG2）的实验结果得到了相同的参数，这些参数描述了 NuG2 的展开和折叠动力学，这表明在力谱实验中拉伸多蛋白 NuG2 确实等效于拉伸单个 NuG2，从而验证了在 SMFS 实验中使用多蛋白的合理性。
