Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

• Published in: Angewandte Chemie International Edition Vol. 58; no. 9; pp. 2730 - 2733
• Main Authors: Chen, Xiaomin, Yao, Haijie, Wang, Hui, Mao, Yunyun, Liu, Dan, Long, Dong
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 25-02-2019
• Edition: International ed. in English
• Subjects: active Ras; Allosteric properties; chemical exchange saturation transfer; Guanosine diphosphate; Guanosine triphosphate; Inhibitors; native GTP; NMR; NMR spectroscopy; Nuclear magnetic resonance; relaxation dispersion; Stability; native GTP; relaxation dispersion; NMR spectroscopy; active Ras; chemical exchange saturation transfer
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201812902

Characterization of native GTP‐bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras⋅GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras⋅GDP signals, is applied to the site‐resolved NMR measurement of the allosteric dynamics of Ras⋅GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor‐binding pockets, but the motions are more confined than those of Ras⋅GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos‐based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.
Concerted motions: An effective approach for the significant extension of the lifetime of active Ras complexed with native GTP is presented, which, along with a postprocessing method that filters out residual Ras⋅GDP signals, permits the accurate quantification of the allosteric dynamics of Ras⋅GTP with site‐specific details by relaxation‐based NMR spectroscopy.