Piconewton-Scale Analysis of Ras-BRaf Signal Transduction with Single-Molecule Force Spectroscopy

Chae-Seok Lim, Cheng Wen, Yanghui Sheng, Guangfu Wang, Zhuan Zhou, Shiqiang Wang, Huaye Zhang, Anpei Ye, J. Julius Zhu

Intermolecular interactions dominate the behavior of signal transduction in various physiological and pathological cell processes, yet assessing these interactions remains a challenging task. Here, this study reports a single-molecule force spectroscopic method that enables functional delineation of two interaction sites (≈35 pN and ≈90 pN) between signaling effectors Ras and BRaf in the canonical mitogen-activated protein kinase (MAPK) pathway. This analysis reveals mutations on BRaf at Q257 and A246, two sites frequently linked to cardio-faciocutaneous syndrome, result in ≈10−30 pN alterations in Ras[BOND]BRaf intermolecular binding force. The magnitude of changes in Ras[BOND]BRaf binding force correlates with the size of alterations in protein affinity and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-sensitive glutamate receptor (-R)-mediated synaptic transmission in neurons expressing replacement BRaf mutants, and predicts the extent of learning impairments in animals expressing replacement BRaf mutants. These results establish single-molecule force spectroscopy as an effective platform for evaluating the piconewton-level interaction of signaling molecules and predicting the behavior outcome of signal transduction.

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