Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants.

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 its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance 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 wild type versus mutants and may thus guide new strategies for the development of therapeutics.

Quantitative Multistate Binding Model of Silica Nanoparticle-Protein Interactions Obtained from Multinuclear Spin Relaxation.

Nanoparticle-assisted NMR spin relaxation (NASR), which makes internal protein dynamics in solution directly observable on nanosecond to microsecond time scales, has been applied to different nuclei and relaxation processes of the same protein system. A model is presented describing the transient interaction between ubiquitin and anionic silica nanoparticles for the unified interpretation of a wealth of experimental data including 2H, 13C, and 15N relaxation of methyl side chain and backbone moieties. The best model, implemented using a stochastic Liouville equation, describes the exchange process via an intermediary encounter state between free and fully nanoparticle-bound protein. The implication of the three-state binding model on the interpretation of NASR data is discussed.

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation.

Amino-acid side-chain properties in proteins are key determinants of protein function. NMR spin relaxation of side chains is an important source of information about local protein dynamics and flexibility. However, traditional solution NMR relaxation methods are most sensitive to sub-nanosecond dynamics lacking information on slower ns-µs time-scale motions. Nanoparticle-assisted NMR spin relaxation (NASR) of methyl-side chains is introduced here as a window into these ns-µs dynamics. NASR utilizes the transient and nonspecific interactions between folded proteins and slowly tumbling spherical nanoparticles (NPs), whereby the increase of the relaxation rates reflects motions on time scales from ps all the way to the overall tumbling correlation time of the NPs ranging from hundreds of ns to µs. The observed motional amplitude of each methyl group can then be expressed by a model-free NASR S2 order parameter. The method is demonstrated for 2H-relaxation of CH2D methyl moieties and cross-correlated relaxation of CH3 groups for proteins Im7 and ubiquitin in the presence of anionic silica-nanoparticles. Both types of relaxation experiments, dominated by either quadrupolar or dipolar interactions, yield highly consistent results. Im7 shows additional dynamics on the intermediate time scales taking place in a functionally important loop, whereas ubiquitin visits the majority of its conformational substates on the sub-ns time scale. These experimental observations are in good agreement with 4-10 µs all-atom molecular dynamics trajectories. NASR probes side-chain dynamics on a much wider range of motional time scales than previously possible, thereby providing new insights into the interplay between protein structure, dynamics, and molecular interactions that govern protein function.

Broadband Dynamics of Ubiquitin by Anionic and Cationic Nanoparticle Assisted NMR Spin Relaxation.

The quantitative and comprehensive description of the internal dynamics of proteins is critical for understanding their function. Nanoparticle-assisted 15 N NMR spin relaxation spectroscopy is a new method for the observation of picosecond to microsecond dynamics of proteins when transiently interacting with the surface of the nanoparticles (NPs). The method is applied here to the protein ubiquitin in the presence of anionic and cationic silica NPs (SNPs) of different sizes. The backbone dynamics profiles are reproducible and strikingly similar to each other, indicating that specific protein-SNP interactions are unimportant. The dynamics profiles closely match the sub-nanosecond dynamics S2 values observed by model-free analysis of standard 15 N relaxation of ubiquitin in free solution, indicating that the bulk of the ubiquitin backbone dynamics in solution is confined to sub-nanosecond timescales and, hence, it is dynamically more restrained than previous NMR studies have suggested.

Functional protein dynamics on uncharted time scales detected by nanoparticle-assisted NMR spin relaxation.

Protein function depends critically on intrinsic internal dynamics, which is manifested in distinct ways, such as loop motions that regulate protein recognition and catalysis. Under physiological conditions, dynamic processes occur on a wide range of time scales from subpicoseconds to seconds. Commonly used NMR spin relaxation in solution provides valuable information on very fast and slow motions but is insensitive to the intermediate nanosecond to microsecond range that exceeds the protein tumbling correlation time. Presently, very little is known about the nature and functional role of these motions. It is demonstrated here how transverse spin relaxation becomes exquisitely sensitive to these motions at atomic resolution when studying proteins in the presence of nanoparticles. Application of this novel cross-disciplinary approach reveals large-scale dynamics of loops involved in functionally critical protein-protein interactions and protein-calcium ion recognition that were previously unobservable.