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KRAS4B is a membrane-anchored signaling protein and a primary target in cancer research. Predictions from molecular dynamics simulations that have previously shaped our mechanistic understanding of KRAS signaling disagree with recent experimental results from neutron reflectometry, NMR, and thermodynamic binding studies. To gain insight into these discrepancies, we compare this body of biophysical data to back-calculated experimental results from a series of molecular simulations that implement different subsets of molecular interactions. Our results show that KRAS4B approximates an entropic ensemble of configurations at model membranes containing 30% phosphatidylserine lipids, which is not significantly shaped by interactions between the globular G-domain of KRAS4B and the lipid membrane. These findings revise our understanding of KRAS signaling and promote a model in which the protein samples the accessible conformational space in a near-uniform manner while being available to bind to effector proteins.

Graphic Abstract Small GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms. Supplementary Information The online version contains supplementary material available at 10.1007/s00232-021-00176-z.

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein–protein and protein–lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.

Second harmonic generation (SHG) is an emergent biophysical method that sensitively measures real-time conformational change of biomolecules in the presence of biological ligands and small molecules. This study describes the successful implementation of SHG as a primary screening platform to identify fragment ligands to oncogenic Kirsten rat sarcoma (KRas). KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a fragment binder to KRas(G12D) and used (1)H (15)N transverse relaxation optimized spectroscopy (TROSY) heteronuclear single-quantum coherence (HSQC) NMR to characterize its binding site as a pocket adjacent to the switch 2 region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared with 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI), a Ras ligand previously described to bind the same pocket. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which is consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.

The development and progression of many human diseases often result in changes in gene expression and protein and metabolite concentrations. Changes at the protein and metabolite level often are detectable in biological fluids and tissues before the appearance of clinical symptoms, rendering them useful diagnostic and prognostic biomarkers. As with many conditions, the discovery of a sensitive and specific urinary biomarker for bladder cancer would save lives and reduce the suffering due to this condition. A number of potential urinary protein biomarkers for bladder cancer have been identified, but they lack the sensitivity and specificity required to replace cystoscopy and histopathology. We discuss the use of mass spectrometry and nuclear magnetic resonance spectroscopy for the detection of metabolites in biological samples, comment on their advantages and limitations, and discuss recently published work in urine metabolic profiling for bladder cancer detection.

High-resolution, liquid state nuclear magnetic resonance (NMR) spectroscopy is a popular platform for metabolic profiling because the technique is nondestructive, quantitative, reproducible, and the spectra contain a wealth of biochemical information. Because of the large dynamic range of metabolite concentrations in biofluids, statistical analyses of one-dimensional (1D) proton NMR data tend to be biased toward selecting changes in more abundant metabolites. Although two-dimensional (2D) proton-proton experiments can alleviate spectral crowding, they have been mainly used for structural determination. In this study, 2D total correlation spectroscopy NMR was used to compare the global metabolic profiles of urine obtained from wild-type and Abcc6-knockout mice. The 2D data were compared to an improved 1D experiment in which signal contributions from macromolecules and the urea peak have been spectroscopically removed for more accurate quantitation of low-abundance metabolites. Although statistical models from both 1D and 2D data could differentiate samples acquired from the two groups of mice, only the 2D spectra allowed the characterization of statistically relevant changes in the low-abundance metabolites. While acquisition of the 2D data require more time, the data obtained resulted in a more meaningful and comprehensive metabolic profile, aided in metabolite identifications, and minimized ambiguities in peak assignments.

The biophysical properties of oligodeoxyribonucleotides (ODNs) selectively modified with conformationally 'locked' bicyclo[3.1.0]hexane pseudosugars (Maier,M.A., Choi,Y., Gaus,H., Barchi,J.J. Jr, Marquez,V.E., Manoharan,M. (2004) Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs Nucleic Acids Res., 32, 3642-3650) have been studied by various techniques. Six separate synthetic ODNs based on the Dickerson Drew dodecamer sequence (CGCGAAT*T*CGCG) were examined where each one (or both) of the thymidines (T*) were substituted with a bicyclic pseudosugar locked in either a North (2'-exo) or South (3'-exo) ring pucker. Circular dichroism spectroscopy, differential scanning calorimetry and (1)H NMR spectroscopy were used to examine the duplex stability and conformational properties of the ODNs. Replacement of one or both thymidines with North-locked sugars (RNA-like) into the dodecamer did not greatly affect duplex formation or melt temperatures but distinct differences in thermodynamic parameters were observed. In contrast, incorporation of South-locked sugar derivatives that were predicted to stabilize this standard B-DNA, had the unexpected effect of causing a conformational equilibrium between different duplex forms at specific strand and salt concentrations. Our data and those of others suggest that although DNA can tolerate modifications with RNA-like (North) nucleotides, a more complicated spectrum of changes emerges with modifications restricted to South (DNA-like) puckers.

The analysis of metabolic pathways for dysfunction has been used for many years in the scientific and medical community to determine overall health. Metabonomics (metabolomics), the global profiling of metabolites, has experienced a rekindling of interest due, in part, to advances in analytical instrumentation for conducting measurements and informatics available for interpretation of the data acquired in this area of biomedical research. Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) based approaches are two primary analytical methods of choice for conducting metabonomic measurements. To overcome the complexity and wide dynamic range of concentrations of metabolites present in biological samples, a common practice is to couple online an analytical separation, typically high performance liquid chromatography (HPLC), with the mass spectrometer. Hence, of critical importance are not only the MS acquisition parameters, but also optimization of those variables that impact th...

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool capable of providing a comprehensive metabolic profile of biofluids such as urine, plasma, and serum. Unfortunately, when measuring serum and plasma, the high protein concentration can obscure the signals originating from low molecular weight metabolites. We evaluated the use of different parameters within the Carr–Purcell–Meiboom–Gill (CPMG) pulse train of fast spin-echoes to remove the macromolecular signal contribution in one-dimensional proton ( 1 H ) NMR spectra. Experimental parameters such as the refocusing delay in the CPMG pulse train, pulse miscalibration, and recycle time were examined to assess the ability to remove the protein signals from the spectrum without causing a deleterious effect on the signals originating from free, low molecular weight metabolites. The 1 H -NMR spectra of a variety of serum samples spiked with 2′-deoxyadenosine were acquired using various acquisition parameters. Our results show that the delay used in the CPMG spin-echo and the combination of the acquisition pulse flip angle and recycle time are the two major factors affecting the observed metabolite signal amplitudes in the resulting 1 H -NMR spectrum.

Summary Turnip crinkle virus encodes two proteins, p8 and p9, that are both required for cell-to-cell movement. The p8 movement protein has been demonstrated to bind RNA in a cooperative manner, although, similar to many other plant virus movement proteins, it contains no canonical RNA binding domain(s). However, three positively charged regions of p8 may potentially form ionic interactions with the RNA backbone. To identify functional regions of p8, a series of alanine and deletion scanning mutations were produced. The effects of these mutations were analysed using both in vitro RNA binding assays and in vivo infections of susceptible (Di-3) and resistant (Di-17) Arabidopsis thaliana plants. Several mutants that have reduced RNA binding ability were also demonstrated to be movement deficient and replication competent. Based on these results, there appear to be two regions, located between amino acids 18 and 31, and 50 and 72, that are required for RNA binding. Furthermore, additional regions (amino acids 12-15, and 34-37) appear to play a role in vivo unrelated to in vitro RNA binding activity.

The 19F19F nuclear spin−spin coupling constants JFF for a set of eighteen compounds related structurally to 1,8-difluoronaphthalene were measured by 19F NMR spectroscopy. The FF distances dFF in these compounds were determined by ab initio 3-21G* molecular orbital calculations. Consistent with the lone-pair overlap theory of the origins of through-space 19F19F coupling, an exponential relationship is found between JFF and dFF (regression coefficient r2 = 0.991), and a linear relationship is found between JFF and the extent of the overlap interaction between the in-plane fluorine 2plone-pair orbitals (regression coefficient r2 = 0.993). The magnitudes of these lone-pair interactions were estimated from molecular orbital energies obtained by ab initio 6-31G* calculations for a model consisting of a pair of HF molecules separated by various distances.

A new method to increase the sensitivity of the 1D transient NOE experiment for molecules in the positive NOE regime is presented. This method, the reverse NOE, simply replaces the conventional relaxation delay between scans. Transient positive NOE enhancements from all other spins are used to accelerate the recovery of the target resonance toward its equilibrium intensity. In favorable cases, the intensity of the target peak at the start of an experiment can actually be increased beyond its equilibrium value. There is also a sensitivity enhancement in the rapid pulsing regime, where recovery is always incomplete. This sensitivity enhancement is illustrated with the one-dimensional double pulsed field gradient spin echo NOE experiment to observe a "fourth-order" NOE. Sensitivity gains of 30% are demonstrated.

Covalent crosslinks can control local peptide conformation. In tripeptide sequences of the general formula Cys-Xxx-Cys, cysteine disulfides have been previously shown to enforce a C7 equatorial gamma-turn conformation (also referred to as an inverse gamma-turn). Much less is known about the effects of dityrosine and ditryptophan crosslinks on local peptide structure. In a series of tripeptides, ditryptophan crosslinks were formed using the two-step process of acid-promoted Mannich dimerization followed by oxidative aromatization. In these peptides, with the general formula Trp-Xxx-Trp (Xxx not equal to Gly), ditryptophan crosslinks were found to stabilize a C7 equatorial gamma-turn conformation in DMSO-d6. Rigorous support for a C7 equatorial conformation in the crosslinked sequence Trp-Pro-Trp came from a variety of 1H NMR experiments and molecular modelling. Interproton distances were derived from NOE buildups that were determined through a series of double pulsed field gradient spin echo (DPFGSE) experiments. In addition, the small temperature dependence of the i+2 NH chemical shifts (delta delta/delta T2 ppm/degree C) provided further support for the intramolecular hydrogen bond which defines a gamma-turn.

A tetraanionic molecular square containing four pentacovalent siliconates is produced in high yield by the condensation of phenyltriethoxysilane and spirocatechol 1. A single isomer, meso 2, is produced from this diastereoselective condensation reaction. The steps leading up to the formation of meso-2 are not known, but the reaction appears to be driven by the insolubility of the tetrakistriethylammonium salt 2a. The tetraanionic macrocycle is a stable salt that undergoes ion-exchange reactions. An ion pair composed of the tetraanionic siliconate macrocycle and cyclobis(paraquat-p-phenylene) tetracation (2e) has been synthesized by ion exchange. Following complete assignment of all proton resonances in this 4 × 4 complex, the solution conformation was analyzed by an intermolecular double pulsed field gradient spin−echo (DPFGSE) NOE study. These results, aided by a molecular mechanics modeling study, have led to a proposed dynamic solution structure that features the cyclic bisparaquat tetracation selectiv...

Previously, it has been shown that the use of pulsed field gradients in one-dimensional NOE experiments results in spectra of much higher quality than it has previously been possible to record. Such high-quality spectra make it possible to measure, with complete confidence, very small NOE enhancements and also make it straightforward to measure NOE buildup curves. In this paper, the spin dynamics behind these NOE experiments with gradients are analyzed in detail; the effects of strong coupling and molecular diffusion are also examined. It is also shown how the basic experiments can be improved upon, in particular how unwanted anti-phase contributions (SPT effects) can be eliminated entirely. Experimental illustrations of the various methods and improvements are given and practical recommendations made as to how the experiments are best performed.

The viability of nuclear magnetic resonance (NMR) spectroscopy as a tool for biomarker discovery has increased over the years as improvements in instrumentation that address NMR's inherent low sensitivity and bioinformatics resources for NMR data handling and analysis have matured. In this chapter, current NMR strategies for metabolic profiling are surveyed. The main focus is analysis of small molecules in biological samples using high-resolution liquid-state techniques. Analysis of cell and tissue via ex vivo and in vivo methods is also discussed. The methodologies covered include one- and two-dimensional experiments, as well as sample preparation and data treatment for statistical analysis.

Demand for the experimental antineoplastic agent schweinfurthin A, for developmental testing, prompted a re-collection of leaf material of Macaranga schweinfurthii from the original collection site in Cameroon. During chromatographic purification of the organic solvent extract, analytical UPLC-PDA-TOFMS of stilbene-enriched fractions revealed the presence of six known schweinfurthins and two previously unknown stilbenes. The structures of these new compounds, schweinfurthins I and J (1 and 2), were elucidated by 1D- and 2D-NMR techniques.

Metabolomics, the global profiling of metabolites in different living systems, has experienced a rekindling of interest partially due to the improved detection capabilities of the instrumental techniques currently being used in this area of biomedical research. The analytical methods of choice for the analysis of metabolites in search of disease biomarkers in biological specimens, and for the study of various low molecular weight metabolic pathways include NMR spectroscopy, GC/MS, CE/MS, and HPLC/MS. Global metabolite analysis and profiling of two different sets of data results in a plethora of data that is difficult to manage or interpret manually because of their subtle differences. Multivariate statistical methods and pattern-recognition programs were developed to handle the acquired data and to search for the discriminating features between data acquired from two sample sets, healthy and diseased. Metabolomics have been used in toxicology, plant physiology, and biomedical research. In this paper, we discuss various aspects of metabolomic research including sample collection, handling, storage, requirements for sample analysis, peak alignment, data interpretation using statistical approaches, metabolite identification, and finally recommendations for successful analysis.

4'-C-ethynyl-2'-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To address this problem, we removed the 3'-OH group from 4'-C-ethynyl-2'-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3'-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4'-C-ethynyl-2',3'-dideoxycytidine analogue (rac-1h) was obtained after 13 steps. In a cellular assay, rac-1h was completely inactive (0.001-10 microM) against HIV(LAI), demonstrating the critical importance of the 3'-OH for antiviral activity. To determine whether the role of the 3'-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5'-triphosphate (rac-1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac-1h-TP was slightly more potent than AZT-5'-triphosphate against wild-type HIV RT, suggesting that the role of the 3'-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac-1h into ent-1h (beta-D-dideoxyribo) and ent-14 (beta-L-dideoxyribo) and the synthesis of the corresponding 5'-triphosphates established the stereochemical assignment based on HIV RT's preference for the beta-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the D-configuration.

Poecillastrin A (1), a new polyketide-derived macrolide lactam, was isolated from a deep-water collection of the marine sponge Poecillastra species. The structure of poecillastrin A (1) was assigned using NMR data acquired at 500 MHz with an inverse-detection cryogenic probe and at 800 MHz with a room-temperature probe.

In two-dimensional homonuclear experiments, reliable detection of very small cross peaks is sometimes hampered by t1-noise from larger diagonal peaks which have the same F2 frequency. This t1-noise can be selectively removed by recording partial 2D spectra using the double pulsed field gradient spin echo (DPFGSE). Resonances not selected by the DPFGSE do not contribute any t1-noise to the resulting spectrum. The absence of t1-noise from the diagonal peaks render the small cross peaks at the same F2 frequency observable. The success of this technique is shown by partial 2D NOESY and TOCSY spectra.