DOI: 10.5061/dryad.j6q573nm0
title: Excited state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

description: Despite the prominent role of the K-Ras protein in many different types of
 human cancer, major gaps in atomic-level information severely limit our
 understanding of K-Ras function in health and disease. Here, we report the
 quantitative backbone structural dynamics of K-Ras by solution NMR
 spectroscopy of the active state of wild-type K-Ras·GTP and two of its
 oncogenic P-loop mutants, G12D and G12C, using a novel
 nanoparticle-assisted spin relaxation method, relaxation dispersion and
 chemical exchange saturation transfer experiments covering the entire
 range of timescales from picosecond to milliseconds. Our combined
 experiments allow the detection and analysis of the functionally critical
 Switch I and Switch II regions that have previously remained largely
 unobservable by X-ray crystallography and NMR spectroscopy. Our data
 reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited
 state that closely resembles the partially disordered K-Ras·GDP state.
 These results advance our understanding of differential GTPase activities
 and signaling properties of the WT versus mutants and may thus guide new
 strategies for the development of therapeutics.
description: NMR relaxation dispersion experiments were acquired on an 850 MHz
 Bruker magnet equipped with a 5 mm TCI cryoprobe and a 600 MHz Bruker
 magnet equipped with a 5 mm TXI cryoprobe at 298 K. Amide
 <sup>15</sup>N CPMG experiments were acquired at both magnetic
 fields using either the CW-CPMG or STCW-CPMG pulse sequences. The constant
 relaxation time was set to 40 ms and the CPMG pulsing frequency,
 <em>ν<sub>CPMG</sub></em>, was varied from 25 Hz
 to 2 kHz on the 850 MHz instrument, and from 25 Hz to 1 kHz on the 600 MHz
 instrument. Amide <sup>1</sup>H<sup>N</sup> CPMG
 experiments were acquired using the sequence of Yuwen and Kay. A constant
 relaxation time of 16 ms was used and νCPMG was varied from 62.5 Hz to 4
 kHz at the 850 MHz instrument only. Amide <sup>15</sup>N
 CEST experiments were performed for all samples on the 850 MHz instrument
 using a CEST mixing time of 150 ms and B1 field strengths of ~40 Hz.
 Additional amide HSQC/HMQC experiments were acquired at both 600 and 850
 MHz to aid in the sign determination of small <sup>15</sup>N
 CPMG-derived chemical shift differences improving the robustness of the
 global fitting procedure.  Backbone
 amide <sup>15</sup>N R<sub>1</sub> and
 R<sub>2</sub> spin relaxation rates for samples both in the
 absence and presence of SNPs were measured at 850 MHz NMR magnetic field
 strength using
 standard <sup>15</sup>N R<sub>1</sub> and
 R<sub>1ρ</sub> relaxation experiments. The recovery delays
 were set to 2 s, and the R<sub>1ρ</sub> spinlock field
 strength was around 2000 Hz. Peak fitting errors were estimated from
 replicate delays in the measurements and propagated by analytical error
 propagation or Monte Carlo simulations through the analysis.
description: All relaxation dispersion data are provided as text files that
 can be used by the ChemEx software. A table of
 relaxation rates and S<sup>2</sup> values is provided for NASR
 results.