Conserved allosteric perturbation of the GTPase domains by region 1 of Ras hypervariable regions.

Ras proteins are important intracellular signaling hubs that can interact with numerous downstream effectors and upstream regulators through their GTPase domains (G-domains) anchored to plasma membranes by the C-terminal hypervariable regions (HVRs). The biological functions of Ras were proposed to be regulated at multiple levels including the intramolecular G-domain-HVR interactions, of which the exact mechanism and specificity are still controversial. Here, we demonstrate that the HVRs, instead of having direct contacts, can weakly perturb the G-domains via an allosteric interaction that is restricted to a ∼20 Å range and highly conserved in the tested Ras isoforms (HRas and KRas4B) and nucleotide-bound states.

Experimental Study on Enhanced Oil Recovery Effect of Profile Control System-Assisted Steam Flooding.

Steam flooding is an effective development method for heavy oil reservoirs, and the steam flooding assisted by the profile control system can plug the dominant channels and further improve the recovery factor. High-temperature-resistant foam as a profile control system is a hot research topic, and the key lies in the optimal design of the foam system. In this paper, lignin was modified by sulfonation to obtain a high-temperature-resistant modified lignin named CRF; the foaming agent CX-5 was confirmed to have good high-temperature foaming ability by reducing the surface tension; the formula of the profile control system (A compound system of CRF and CX-5, abbreviated as PCS) and the best application parameters were optimized by the foam resistance factor.

Quantitative Ensemble Interpretation of Membrane Paramagnetic Relaxation Enhancement (mPRE) for Studying Membrane-Associated Intrinsically Disordered Proteins.

An understanding of the functional role played by a membrane-associated intrinsically disordered protein (IDP) requires characterization of its heterogeneous conformations as well as its poses relative to the membranes, which is of great interest but technically challenging. Here, we explore the membrane paramagnetic relaxation enhancement (mPRE) for constructing ensembles of IDPs that dynamically associate with membrane mimetics incorporating spin-labeled lipids. To accurately interpret the mPRE Γ rates, both the dynamics of IDPs and spin probe molecules are taken into account, with the latter described by a weighted three-dimensional (3D) grid model built based on all-atom simulations.

Quantitative Paramagnetic NMR-Based Analysis of Protein Orientational Dynamics on Membranes: Dissecting the KRas4B-Membrane Interactions.

Peripheral membrane proteins can adopt distinct orientations on the surfaces of lipid bilayers that are often short-lived and challenging to characterize by conventional experimental methods. Here we describe a robust approach for mapping protein orientational landscapes through quantitative interpretation of paramagnetic relaxation enhancement (PRE) data arising from membrane mimetics with spin-labeled lipids. Theoretical analysis, followed by experimental verification, reveals insights into the distinct properties of the PRE observables that are generally distorted in the case of stably membrane-anchored proteins.

Exploring the state- and allele-specific conformational landscapes of Ras: understanding their respective druggabilities.

Recent advances in direct inhibition of Ras benefit from the protein's intrinsic dynamic nature that derives therapeutically vulnerable conformers bearing transiently formed cryptic pockets. Hotspot mutants of Ras are major tumor drivers and are hyperactivated in cells at variable levels, which may require allele-specific strategies for effective targeting. However, it remains unclear how the prevalent oncogenic mutations and activation states perturb the free energy landscape governing the protein dynamics and druggability.

A First-Principles Study on the Hydration Behavior of (MgO) Clusters and the Effect Mechanism of Anti-Hydration Agents.

Magnesia-based refractory is widely used in high-temperature industries; its easy hydration is, however, a key concern in refractory processing. Understanding the hydration mechanism of MgO will help in solving its hydration problem. Herein, the hydration behavior of (MgO) (n = 1-6) at the molecular level and the effect mechanisms of several anti-hydration agents on the hydration of (MgO) were investigated with first-principles calculations.

Unveiling the "invisible" druggable conformations of GDP-bound inactive Ras.

The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale.

Cross-Species Comparison of Metabolomics to Decipher the Metabolic Diversity in Ten Fruits.

Fruits provide humans with multiple kinds of nutrients and protect humans against worldwide nutritional deficiency. Therefore, it is essential to understand the nutrient composition of various fruits in depth. In this study, we performed LC-MS-based non-targeted metabolomic analyses with ten kinds of fruit, including passion fruit, mango, starfruit, mangosteen, guava, mandarin orange, grape, apple, blueberry, and strawberry.

Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue.

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP.

Combined Analysis of Transcriptome and Metabolome Reveals the Potential Mechanism of Coloration and Fruit Quality in Yellow and Purple Sims.

Passion fruit ( Sims) can be divided into yellow and purple varieties. However, information about coloration and fruit quality between the two varieties is limited. To reveal the underlying mechanism of color formation in this fruit, a combined analysis of the metabolome and transcriptome was conducted in this study.

and Mediate the Mild Osmotic Stress-Induced Root Growth Response in .

Although most osmotic stresses are harmful to plant growth and development, certain drought- or polyethylene glycol (PEG)-induced mild osmotic stresses promote plant root growth. The underlying regulatory mechanisms of this response remain elusive. Here, we report that the GLYCOGEN SYNTHASE KINASE 3 (GSK3) genes () ) and () are involved in the mild osmotic stress (-0.

NMR-Derived Conformational Ensemble of State 1 of Activated Ras Reveals Insights into a Druggable Pocket.

The lack of apparent pockets in the ground conformation of Ras has long challenged the rational design of inhibitors against this oncogenic protein. The sparsely populated, transiently formed state 1 of activated Ras, on the other hand, shows appreciable surface roughness and is increasingly recognized as a potential target for drug discovery. State 1, however, is extremely flexible, and a static structure cannot fully unveil its conformational space that can be exploited for drug design.

Atomistic Insights into the Functional Instability of the Second Helix of Fatty Acid Binding Protein.

Structural dynamics of fatty acid binding proteins (FABPs), which accommodate poorly soluble ligands in the internalized binding cavities, are intimately related to their function. Recently, local unfolding of the α-helical cap in a variant of human intestinal FABP (IFABP) has been shown to correlate with the kinetics of ligand association, shedding light on the nature of the critical conformational reorganization. Yet, the physical origin and mechanism of the functionally relevant transient unfolding remain elusive.

Effects of rumen cannulation on dissolved gases and methanogen community in dairy cows.

Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows.

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

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.

Nitrate improves ammonia incorporation into rumen microbial protein in lactating dairy cows fed a low-protein diet.

Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control.

Characterization of the interaction interface and conformational dynamics of human TGIF1 homeodomain upon the binding of consensus DNA.

The TG interacting factor-1 homeodomain (TGIF1-HD) binds with the consensus DNA motif 5'-TGTCA-3' in gene promoters through its three-amino acid loop extension (TALE) type homeodomain, and then recruits co-regulators to regulate gene expression. Although the solution NMR structure of human TGIF1-HD has been reported previously, little is known about its DNA binding mechanism. NMR titrations have been extensively used to study mechanisms of ligand binding to target proteins; however, an intermediate exchange occurred predominantly between TGIF1-HD in the free and bound states when titrated with the consensus DNA, which resulted in poor-quality NMR spectra and precluded further exploration of its interaction interface and conformational dynamics.

Probing Transient Release of Membrane-Sequestered Tyrosine-Based Signaling Motif by Solution NMR Spectroscopy.

Membrane sequestration of tyrosine-based signaling motifs of antigen receptors effectively restricts the signaling activities in resting lymphocytes. However, low level of basal signaling in resting cells is required for lymphocyte survival and antigen responsiveness, of which the molecular mechanism remains obscure. Here we probe the transient release of the cytoplasmic domain of the membrane-bound IgG heavy chain (mIgG-tail) by hydrogen exchange NMR spectroscopy, illustrating a dynamic molecular basis for its basal signaling activity.

Microsecond Timescale Dynamics of GDP-Bound Ras Underlies the Formation of Novel Inhibitor-Binding Pockets.

The recent discovery of inhibitory compounds binding to distinct pockets on GDP-bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras⋅GDP on microsecond to millisecond timescales using relaxation-based NMR experiments, and identified substantial slow dynamics with τ of 34 μs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor-bound and free states.

Diversity and Composition of Bacterial Community in Soils and Lake Sediments from an Arctic Lake Area.

This study assessed the diversity and composition of bacterial communities within soils and lake sediments from an Arctic lake area (London Island, Svalbard). A total of 2,987 operational taxonomic units were identified by high-throughput sequencing, targeting bacterial 16S rRNA gene. The samples from four sites (three samples in each site) were significantly different in geochemical properties and bacterial community composition.

Probing Invisible, Excited Protein States by Non-Uniformly Sampled Pseudo-4D CEST Spectroscopy.

Chemical exchange saturation transfer (CEST) NMR spectroscopy is a powerful tool for studies of slow timescale protein dynamics. Typical experiments are based on recording a large number of 2D data sets and quantifying peak intensities in each of the resulting planes. A weakness of the method is that peaks must be resolved in 2D spectra, limiting applications to relatively small proteins.

Triple resonance-based ¹³C(α) and ¹³C(β) CEST experiments for studies of ms timescale dynamics in proteins.

A pair of triple resonance based CEST pulse schemes are presented for measuring ¹³C(α) and ¹³C(β) chemical shifts of sparsely populated and transiently formed conformers that are invisible to traditional NMR experiments. CEST profiles containing dips at resonance positions of ¹³C(α) or ¹³C(β) spins of major (ground) and minor (excited) conformers are obtained in a pseudo 3rd dimension that is generated by quantifying modulations of cross peaks in ¹⁵N, ¹H(N) correlation spectra. An application to the folding reaction of a G48A mutant of the Fyn SH3 domain is presented, illustrating and validating the methodology.

Measuring hydrogen exchange rates in invisible protein excited states.

Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct (15)N chemical shifts.

Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica.

The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response.