3 years ago

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

Piconewton-Scale Analysis of Ras-BRaf Signal Transduction with Single-Molecule Force Spectroscopy
Huaye Zhang, J. Julius Zhu, Cheng Wen, Chae-Seok Lim, Guangfu Wang, Anpei Ye, Shiqiang Wang, Zhuan Zhou, Yanghui Sheng
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 RasBRaf intermolecular binding force. The magnitude of changes in RasBRaf 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. Intermolecular interactions define signal transduction behavior, yet assessing these interactions remains a daunting task. Here, a single-molecule force spectroscopic method is adpated to funcationally delineate two (≈35 pN and ≈90 pN) interaction sites between signaling effectors Ras and BRaf in the canonical MAPK pathway, and this piconewton analysis approach is independently validated with biochemical, electrophysiological, and behavioral measurements.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/smll.201701972

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