Solution-state NMR Spectroscopy Research Articles

Structural Characterization of 6-Halo-6-Deoxycelluloses by Direct-Dissolution Solution-State NMR Spectroscopy.

Regioselective modifications of cellulose using activated cellulose derivatives such as 6-halo-6-deoxycelluloses provide a convenient approach for developing sustainable products with properties tailored to specific applications. However, maintaining precise regiochemical control of substituent distribution in 6-halo-6-deoxycelluloses is challenging due to their insolubility in most common solvents and the resulting difficulties in precise structure elucidation by modern instrumental analytical techniques. Herein, an accessible NMR-based approach toward detailed characterization of 6-halo-6-deoxycelluloses, including the determination of the degrees of substitution at carbon 6 (DS6), is presented. It is shown that the direct-dissolution cellulose solvent, tetrabutylphosphonium acetate:DMSO-d6, converts 6-halo-6-deoxycelluloses to 6-monoacetylcellulose, enabling in situ solution-state NMR measurements. Arange of 1D and 2D NMR experiments is used to demonstrate the quantitivity of the conversion and provide optimum dissolution conditions. In comparison with other NMR-based derivatization protocols for elucidating the structure of 6-halo-6-deoxycelluloses, the presented approach offers major advantages in terms of accuracy, speed, and simplicity of analysis, and minimal requirements for reagents or NMR instrumentation.

6-Strand to Stable 10/12 Helix Conformational Switch by Incorporating Flexible β-hGly in the Homooligomers of Camphor Derived β-Amino Acid: NMR and X-Ray Crystallographic Evidence.

Rational design of unnatural amino acid building blocks capable of stabilizing predictable secondary structures similar to protein fragments is pivotal for foldamer chemistry/catalysis. Here, we introduce novel β-amino acid building blocks: [1S,2R,4R]exoCDA and [1S,2S,4R]endoCDA, derived from the abundantly available R(+)-camphor, which is traditionally known for its medicinal value. Further, we demonstrate that the homooligomers of exoCDA adopt 6-strand conformation, which switches to a robust 10/12-helix simply by inserting flexible β-hGly spacer at alternate positions (1 : 1 β-hGly/exoCDA heterooligomers), as evident by DFT-calculations, solution-state NMR spectroscopy and X-ray crystallography. To the best of our knowledge, this is the first example of crystalline-state structure of left-handed 10/12-mixed helix, that is free from the conventional approach of employing β-amino acids of either alternate chirality or alternate β2/β3 substitutions, to access the 10/12-helix. The results also show that the homooligomers of heterochiral exoCDA don't adopt helical fold, instead exhibit banana-shaped strands, whereas the homodimers of the other diastereomer endoCDA, nucleate 8-membered turns. Furthermore, the homo-exoCDA and hetero-[β-hGly-exoCDA] oligomers are found to exhibit self-association properties with distinct morphological features. Overall, the results offer new possibilties of constructing discrete stable secondary and tertiary structures based on CDAs, which can accommodate flexible residues with desired side-chain substitutions.

Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Conformational Preferences at the Glycosidic Linkage of Saccharides in Solution as Deduced from NMR Experiments and MD Simulations: Comparison to Crystal Structures.

Glycans are central to information content and regulation in biological systems. These carbohydrate molecules are active either as oligo- or polysaccharides, often in the form of glycoconjugates. The monosaccharide entities are joined by glycosidic linkages and stereochemical arrangements are of utmost importance in determining conformation and flexibility of saccharides. The conformational preferences and population distributions at the glycosidic torsion angles φ and ψ have been investigated for O-methyl glycosides of three disaccharides where the substitution takes place at a secondary alcohol, viz., in α-l-Fucp-(1→3)-β-d-Glcp-OMe, α-l-Fucp-(1→3)-α-d-Galp-OMe and α-d-Glcp-(1→4)-α-d-Galp-OMe, corresponding to disaccharide structural elements present in bacterial polysaccharides. Stereochemical differences at or adjacent to the glycosidic linkage were explored by solution state NMR spectroscopy using one-dimensional 1 H,1 H-NOESY NMR experiments to obtain transglycosidic proton-proton distances and one- and two-dimensional heteronuclear NMR experiments to obtain 3 JCH transglycosidic coupling constants related to torsion angles φ and ψ. Computed effective proton-proton distances from molecular dynamics (MD) simulations showed excellent agreement to experimentally derived distances for the α-(1→3)-linked disaccharides and revealed that for the bimodal distribution at the ψ torsion angle for the α-(1→4)-linked disaccharide experiment and simulation were at variance with each other, calling for further force field developments. The MD simulations disclosed a highly intricate inter-residue hydrogen bonding pattern for the α-(1→4)-linked disaccharide, including a nonconventional hydrogen bond between H5' in the glucosyl residue and O3 in the galactosyl residue, supported by a large downfield 1 H NMR chemical shift displacement compared to α-d-Glcp-OMe. Comparison of population distributions of the glycosidic torsion angles φ and ψ in the disaccharide entities to those of corresponding crystal structures highlighted the potential importance of solvation on the preferred conformation.

Contrasting a series of bidentate amido phosphine oxide, sulfide, or selenide ligands and complexes of dimethyl aluminum and indium.

A series of EP(V)-imine phosphine-imine ligands (Dipp-NC(CH3)-(CH2)-PE(Ph2)) (Dipp = 2,6-diisopropylphenyl, E = O, S, Se) and corresponding dimethyl aluminum and indium complexes were prepared and characterized using multi-nuclear NMR spectroscopy and SC-XRD. Solution-state 1H and 31P NMR spectroscopy indicate a mixture of isomers in solution (C6D6, CDCl3, and CD3CN). Solid state structures of both the imine and (E)-enamine isomers of the oxygen-based ligand have been observed. These three ligands (L) were then deprotonated with M(CH3)3 (M = Al or In), with methane loss forming a 6-membered ring via bidentate chelation ((κ2-L)M(CH3)2). The indium/oxide complex also crystallized as a 12-membered ring.

Possibilities and limitations of solution-state NMR spectroscopy to analyze the ligand shell of ultrasmall metal nanoparticles

Unlike larger plasmonic nanoparticles, ultrasmall nanoparticles with a diameter of 1–2 nm can be well analyzed by NMR spectroscopy. This gives deep insight into the nature of the organic ligand shell.

Structure, dynamics, and stability of the smallest and most complex 71 protein knot

Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule.

NMR Structural Study of Syndecan-4 Transmembrane Domain with Cytoplasmic Region

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP2) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP2.

Solution-State 2D NMR Spectroscopy of Mixtures HyperpolarizedUsing Optically Polarized Crystals.

The HYPNOESYS method (Hyperpolarized NOE System), which relies on the dissolution of optically polarized crystals, has recently emerged as a promising approach to enhance the sensitivity of NMR spectroscopy in the solution state. However, HYPNOESYS is a single-shot method that is not generally compatible with multidimensional NMR. Here we show that 2D NMR spectra can be obtained from HYPNOESYS-polarized samples, using single-scan acquisition methods. The approach is illustrated with a mixture of terpene molecules and a benchtop NMR spectrometer, paving the way to a sensitive, information-rich and affordable analytical method.

Simple and User-Friendly Methodology for Crystal Water Determination by Quantitative Proton NMR Spectroscopy in Deuterium Oxide.

In drug research and development, knowledge of the precise structure of an active ingredient is crucial. However, it is equally important to know the water content of the drug molecule, particularly the number of crystal waters present in its structure. Such knowledge ensures the avoidance of drug dosage and formulation errors since the number of water molecules affects the physicochemical and pharmaceutical properties of the molecule. Several methods have been used for crystal water measurements of organic compounds, of which thermogravimetry and crystallography may be the most common ones. To the best of our knowledge, solution-state NMR spectroscopy has not been used for crystal water determination in deuterium oxide. Quantitative NMR (qNMR) method will be presented in the paper with a comparison of single-crystal X-ray diffraction and thermogravimetric analysis results. The qNMR method for water content measurement is straightforward, reproducible, and accurate, including measurement of 1H NMR spectrum before and after the addition of the analyte compound, and the result can be calculated after integration of the reference compound, analyte, and HDO signals using the given equation. In practical terms, there is no need for weighing the samples under study, which makes it simple and is a clear advantage to the current determination methods. In addition, the crystal structures of two model bisphosphonates used herein are reported: that of monopotassium etidronate dihydrate and monosodium zoledronate trihydrate.

Reinvestigating the concurring reactions in early-stage cellulose pyrolysis by solution state NMR spectroscopy

Low temperature pre-treatments increase the char yield during cellulose carbonization. Although this effect is mostly understood on a kinetic basis, the formed chemical structures leave room for scrutiny. In ongoing ambitions to enhance the char yield of bioderived precursors, the thereby occurring chemistry was reinvestigated. A set of isothermal heating protocols ranging from 150° to 250°C was applied to man-made Ioncell® cellulose fibers and to Avicel® PH-101 microcrystalline cellulose. The prepared cellulosic samples were examined by solution state NMR using a tetra-n-butyl phosphonium acetate ([P4444][OAc]): DMSO-d6 (1:4 wt%) electrolyte as dissolving medium. Complementary, IR spectroscopy, size-exclusion chromatography (SEC) and thermogravimetric analysis coupled with mass spectroscopy (TGA-MS) measurements were performed. The NMR spectra evidenced the formation of levoglucosan end capped moieties as being the first and major occurring reaction during low temperature pre-treatments. In contrast to other mechanistic proposals, no signs for carbonyl or alkene functionalities were discernible in the cellulosic material soluble in the NMR electrolyte, even after treatment at 250 °C for several hours. Thermal cross-linking was observed in SEC already at temperatures not known to significantly influence the overall char yield. In the solution state analytics only partial solubility was observed, owing to the formation of a reluctant fraction previously described as “thermostable condensed phase”. This resulted in analytical blind spots and led to discrepancies between NMR results and FTIR spectra in which carbonyl and alkene vibrations were clearly discernible. Those discrepancies might also imply the co-existence of different fractions in the early stages of cellulose pyrolysis.

Conformational Plasticity of Parallel G-Quadruplex─Implications on Duplex-Quadruplex Motifs.

DNA G-quadruplexes are essential motifs in molecular biology performing a wide range of functions enabled by their unique and diverse structures. In this study, we focus on the conformational plasticity of the most abundant and biologically relevant parallel G-quadruplex topology. A multipronged approach of structure survey, solution-state NMR spectroscopy, and molecular dynamics simulations unravels subtle yet essential features of the parallel G-quadruplex topology. Stark differences in flexibility are observed for the nucleotides depending upon their positioning in the tetrad planes that are intricately correlated with the conformational sampling of the propeller loop. Importantly, the terminal nucleotides in the 5'-end versus the 3'-end of the parallel quadruplex display differential dynamics that manifests their ability to accommodate a duplex on either end of the G-quadruplex. The conformational plasticity characterized in this study provides essential cues toward biomolecular processes such as small molecular binding, intermolecular quadruplex stacking, and implications on how a duplex influences the structure of a neighboring quadruplex.

Nuclear Magnetic Resonance Detection of Hydrogen Bond Network in a Proton Pump Rhodopsin RxR and Its Alteration during the Cyclic Photoreaction.

Hydrogen bond formation and deformation are crucial for the structural construction and functional expression of biomolecules. However, direct observation of exchangeable hydrogens, especially for oxygen-bound hydrogens, relevant to hydrogen bonds is challenging for current structural analysis approaches. Using solution-state NMR spectroscopy, this study detected the functionally important exchangeable hydrogens (i.e., Y49-ηOH and Y178-ηOH) involved in the pentagonal hydrogen bond network in the active site of R. xylanophilus rhodopsin (RxR), which functions as a light-driven proton pump. Moreover, utilization of the original light-irradiation NMR approach allowed us to detect and characterize the late photointermediate state (i.e., O-state) of RxR and revealed that hydrogen bonds relevant to Y49 and Y178 are still maintained during the photointermediate state. In contrast, the hydrogen bond between W75-εNH and D205-γCOO- is strengthened and stabilizes the O-state.

Mechanistic insights into the aggregation pathway of the patient-derived immunoglobulin light chain variable domain protein FOR005

Systemic antibody light chain (AL) amyloidosis is characterized by deposition of amyloid fibrils. Prior to fibril formation, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes. We focus on the patient derived λ-III AL variable domain FOR005 which is mutated at five positions with respect to the closest germline protein. Using solution-state NMR spectroscopy, we follow the individual steps involved in protein misfolding from the native to the amyloid fibril state. Unfavorable mutations in the complementary determining regions introduce a strain in the native protein structure which yields partial unfolding. Driven by electrostatic interactions, the protein converts into a high molecular weight, oligomeric, molten globule. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into fibrils. The topology is determined by balanced electrostatic interactions in the fibril core implying a 180° rotational switch of the beta-sheets around the conserved disulfide bond.

NMR spectroscopy-based analysis of gallstones of cancerous and benign gallbladders from different geographical regions of the Indian subcontinent.

Analysis of the chemical composition of gallstones is vital for the etiopathogenesis of gallstone diseases that can ultimately help in the prevention of its formation. In the present study, gallstones from seven different regions of India were analyzed to highlight the major difference in their composition. Also, gallstones of different pathological conditions i.e., benign (chronic cholecystitis, CC) and malignant gallbladder disease (gallbladder cancer GBC) were characterized. The type of polymorphs of cholesterol molecules was also studied to provide insight into the structure of gallstones. 1H solution state NMR spectroscopy 1D experiments were performed on a total of 94 gallstone (GS) samples collected from seven different geographical regions of India. Solid-State NMR spectroscopy 13C cross-polarization magic angle spinning (CPMAS) experiments were done on the 20 CC GS samples and 20 GBC GS samples of two regions. 1H NMR spectra from the solution state NMR of all the stones reveal that cholesterol was a major component of the maximum stones of the north India region while in south Indian regions, GS had very less cholesterol. 13C CPMAS experiments reveal that the quantity of cholesterol was significantly more in the GS of CC in the Lucknow region compared with GBC stones of Lucknow and Chandigarh. Our study also revealed that GS of the Lucknow region of both malignant and benign gallbladder diseases belong to the monohydrate crystalline form of cholesterol while GS of Chandigarh region of both malignant and benign gallbladder diseases exists in both monohydrate crystalline form with the amorphous type and anhydrous form. Gallstones have a complicated and poorly understood etiology. Therefore, it is important to understand the composition of gallstones, which can be found in various forms and clinical conditions. Variations in dietary practices, environmental conditions, and genetic factors may influence and contribute to the formation of GS. Prevention of gallstone formation may help in decreasing the cases of gallbladder cancer.

Elucidation of the folding pathway of a circular permutant of topologically knotted YbeA by tryptophan substitutions

CP74 is an engineered circular permutant of a deep trefoil knotted SpoU-TrmD (SPOUT) RNA methyl transferase protein YbeA from E. coli. We have previously established that the circular permutation unties the knotted topology of YbeA and CP74 forms a domain-swapped dimer with a large dimeric interface of ca. 4600 Å2. To understand the impact of domain-swapping and the newly formed hinge region joining the two folded domains on the folding and stability of CP74, the five equally spaced tryptophan residues were individually substituted into phenylalanine to monitor their conformational and stability changes by a battery of biophysical tools. Far-UV circular dichroism, intrinsic fluorescence, and small-angle X-ray scattering dictated minimal global conformational perturbations to the native structures in the tryptophan variants. The structures of the tryptophan variants also showed the conservation of the domain-swapped ternary structure with the exception that the W72F exhibited significant asymmetry in the α-helix 5. Comparative global thermal and chemical stability analyses indicated the pivotal role of W100 in the folding of CP74 followed by W19 and W72. Solution-state NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry further revealed the accumulation of a native-like intermediate state in which the hinge region made important contributions to maintain the domain-swapped ternary structure of CP74.

Taming the Dichalcogenides: Isolation, Characterization, and Reactivity of Elusive Perselenide, Persulfide, Thioselenide, and Selenosulfide Anions.

Reactive sulfur species (RSS) and reactive selenium species (RSeS) play integral roles in hydrogen sulfide (H2S) and hydrogen selenide (H2Se) biological signaling pathways, and dichalcogenide anions are proposed transient intermediates that facilitate a variety of biochemical transformations. Herein we report the selective synthesis, isolation, spectroscopic and structural characterization, and fundamental reactivity of persulfide (RSS-), perselenide (RSeSe-), thioselenide (RSSe-), and selenosulfide (RSeS-) anions. The isolated chalcogenides do not rely on steric protection for stability and have steric profiles analogous to cysteine (Cys). Simple reduction of S8 or Se by potassium benzyl thiolate (KSBn) or selenolate (KSeBn) in the presence of 18-crown-6 afforded [K(18-crown-6)][BnSS] (1), [K(18-crown-6)][BnSeSe] (2), [K(18-crown-6][BnSSe] (3), and [K(18-crown-6][BnSeS] (4). The chemical structure of each dichalcogenide was confirmed by X-ray crystallography and solution-state 1H, 13C, and 77Se NMR spectroscopy. To advance our understanding of the reactivity of these species, we demonstrated that reduction of 1-4 by PPh3 readily generates E═PPh3 (E: S, Se), and reduction of 1, 3, and 4 by DTT readily produces HE-/H2E. Furthermore, 1-4 react with CN- to produce ECN-, which is consistent with the detoxifying effects of dichalcogenide intermediates in the Rhodanese enzyme. Taken together, this work provides new insights into the inherent structural and reactivity characteristics of dichalcogenides relevant to biology and advances our understanding of the fundamental properties of these reactive anions.

An NMR Study of a 300-kDa AAA+ Unfoldase

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

Distinct growth regimes of α-synuclein amyloid elongation

Addition of amyloid seeds to aggregation-prone monomers allows for amyloid fiber growth (elongation) omitting slow nucleation. We here combine Thioflavin T fluorescence (probing formation of amyloids) and solution-state NMR spectroscopy (probing disappearance of monomers) to assess elongation kinetics of the amyloidogenic protein, α-synuclein, for which aggregation is linked to Parkinson’s disease. We found that both spectroscopic detection methods give similar kinetic results, which can be fitted by applying double exponential decay functions. When the origin of the two-phase behavior was analyzed by mathematical modeling, parallel paths as well as stop-and-go behavior were excluded as possible explanations. Instead, supported by previous theory, the experimental elongation data reveal distinct kinetic regimes that depend on instantaneous monomer concentration. At low monomer concentrations (toward end of experiments), amyloid growth is limited by conformational changes resulting in β-strand alignments. At the higher monomer concentrations (initial time points of experiments), growth occurs rapidly by incorporating monomers that have not successfully completed the conformational search. The presence of a fast disordered elongation regime at high monomer concentrations agrees with coarse-grained simulations and theory but has not been detected experimentally before. Our results may be related to the wide range of amyloid folds observed.

Abstract PR01: Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues

Abstract RAS mutations occur in approximately 20% of human cancers including the majority of pancreatic ductal adenocarcinoma (PDAC), half of colorectal cancers, and a third of all lung cancers. In cases of oncogenic activating RAS mutations, GTP hydrolysis is impaired, and the protein is preferentially held in the active GTP-bound state. When activated, RAS triggers multiple proliferative signaling cascades, including the MAPK/ERK and PI3K pathways, to induce cell growth, division, and differentiation. In solution, RAS exists in two interconverting GTP-bound conformational states, State 1 (inactive) and State 2 (active). State 2 is the active conformation since it binds to RAS Binding Domain (RBD) of effector proteins. Though the State 1 conformation is GTP-bound, it is unable to bind effectors and represents an inactive KRAS conformation. This State 1/State 2 equilibrium is closely coupled to the conformations (γ1 and γ2) exhibited by the γ-phosphate of GTP and its analogues. Solution-state 31P NMR spectroscopy captures and quantifies the γ1 and γ2 peaks representing State 1 and State 2 (Spoerner M et al., JBC 2010). We have initiated solution-state NMR investigation to determine the structural differences between wild-type (WT) and oncogenic mutant KRAS proteins (G12C, G12D, G12V, Q61L, and G13D) when bound to GTP and GppNHp. We quantified the State 1/State 2 equilibrium using 31P NMR. Our results show that GppNHp-bound KRAS proteins harboring oncogenic mutations differentially modulate the State 1/State 2 conformational equilibria resulting in remarkable divergence in conformational populations compared to WT protein; a notion for presence of mutation induced structural plasticity. Much work on the structural biology, biophysics and biochemistry of KRAS proteins is done when bound to the non-hydrolysable GTP analogue, GppNHp. Strikingly, our 31P NMR results show that GTP-bound KRAS elicits significant variations in State 1 and State 2 populations in comparison to GppNHp-bound proteins. These data suggest GppNHp binding induces a significant increase in the State 1 population; an observation that does not translate into GTP-bound KRAS. Our results provide additional insights into the differential binding affinities seen between WT/mutant KRAS and effector proteins (e.g., Hunter JC et al., Mol Cancer Res 2015) as well as interpretation for State 1 conformations in KRAS crystal structures when bound to mostly GppNHp and not GTP. We have also applied this 31P NMR approach to the evaluation of KRAS small molecule inhibitors that shift the population equilibrium to the State 1 (inactive) conformation in the protein-ligand complex. This demonstrates an intriguing therapeutic opportunity and novel mechanism of action that we are further exploring as part of our drug discovery pipeline. Citation Format: Alok K. Sharma, Marcin Dyba, Dominic Esposito, Bin Wang, Pedro J. Beltran, Eli Wallace, Dwight Nissley, Frank McCormick, Anna E. Maciag. Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR01.