Nuclear Overhauser Effect Spectroscopy Data Research Articles

CD28 hinge used in chimeric antigen receptor (CAR) T-cells exhibits local structure and conformational exchange amidst global disorder

T-cell therapies based on chimeric antigen receptor (CAR) targeting of a tumor-specific antigen offer hope for patients with relapsed or refractory cancers. CAR hinge and transmembrane regions link antigen recognition domains to intracellular signal transduction domains. Here, we apply biophysical methods to characterize the structure and dynamic properties of the CD28 CAR hinge (CD28H) used in an FDA-approved CD19 CAR for the treatment of B-lineage leukemia/lymphoma. By using nuclear Overhauser effect spectroscopy (NOESY), which detects even transiently occupied structural motifs, we observed otherwise elusive local structural elements amidst overall disorder in CD28H, including a conformational switch from a native β-strand to a 310-helix and polyproline II helix-like structure. These local structural motifs contribute to an overall loosely formed extended geometry that could be captured by NOESY data. All FDA-approved CARs use prolines in the hinge region, which we find in CD28, and previously in CD8α, isomerize to promote structural plasticity and dynamics. These local structural elements may function in recognition and signaling events and constrain the spacing between the transmembrane and antigen recognition domains. Our study thus demonstrates a method for detecting local and transient structure within intrinsically disordered systems and moreover, our CD28H findings may inform future CAR design.

Automatic structure-based NMR methyl resonance assignment in large proteins

Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28–358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4–1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.

Two New Cerioptins (A‐B) from the Mangrove Ceriops tagal

AbstractTwo novel compounds (1‐2), cerioptins A−B, along with seven known compounds were isolated from the mangrove, Ceriops tagal. These new compounds were characterized as a rare ethyl‐[(2‐allyl‐2‐chloromethyl)‐2H‐1‐benzopyran]‐3‐carboxylate (1) and a novel tetraterpenoid congener of heterodimeric diterpenoid, pimara‐11‐enol‐(16→2′)‐dolabra‐1,4(18),15‐triene (2) by extensive modern spectroscopic 2D NMR [COSY (correlation spectroscopy), NOESY (Nuclear Overhauser Effect Spectroscopy), HSQC (heteronuclear single‐quantum correlation), HMBC (heteronuclear multiple bond correlation)], IR (infrared) and mass data analysis. The naturally occurring new heterodimeric diterpene is derived from two new diterpenes as pimara‐11‐enol and dolabra‐1,4(18),15‐triene.

A Bayesian Approach for Determining Protein Side-Chain Rotamer Conformations Using Unassigned NOE Data

A major bottleneck in protein structure determination via nuclear magnetic resonance (NMR) is the lengthy and laborious process of assigning resonances and nuclear Overhauser effect (NOE) cross peaks. Recent studies have shown that accurate backbone folds can be determined using sparse NMR data, such as residual dipolar couplings (RDCs) or backbone chemical shifts. This opens a question of whether we can also determine the accurate protein side-chain conformations using sparse or unassigned NMR data. We attack this question by using unassigned nuclear Overhauser effect spectroscopy (NOESY) data, which records the through-space dipolar interactions between protons nearby in three-dimensional (3D) space. We propose a Bayesian approach with a Markov random field (MRF) model to integrate the likelihood function derived from observed experimental data, with prior information (i.e., empirical molecular mechanics energies) about the protein structures. We unify the side-chain structure prediction problem with the side-chain structure determination problem using unassigned NMR data, and apply the deterministic dead-end elimination (DEE) and A* search algorithms to provably find the global optimum solution that maximizes the posterior probability. We employ a Hausdorff-based measure to derive the likelihood of a rotamer or a pairwise rotamer interaction from unassigned NOESY data. In addition, we apply a systematic and rigorous approach to estimate the experimental noise in NMR data, which also determines the weighting factor of the data term in the scoring function derived from the Bayesian framework. We tested our approach on real NMR data of three proteins: the FF Domain 2 of human transcription elongation factor CA150 (FF2), the B1 domain of Protein G (GB1), and human ubiquitin. The promising results indicate that our algorithm can be applied in high-resolution protein structure determination. Since our approach does not require any NOE assignment, it can accelerate the NMR structure determination process.

Spectroscopic Characterization of Molecular Aggregates in Solutions: Impact on Crystallization of Indomethacin Polymorphs from Acetonitrile and Ethanol

Infrared (IR), Raman, and nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the nature of molecular aggregation of indomethacin (I) in acetonitrile and ethanol solutions. Spectroscopic data provided no evidence for self-aggregation of I in supersaturated solutions of acetonitrile or ethanol. As the concentration of I was increased by more than 130 fold, from 5 × 10–4 to 0.067 M in acetonitrile, the diffusion coefficients of I and water (residual) measured by pulse field gradient spin echo nuclear magnetic resonance (PFGSE-NMR) showed a decrease of about 5 and 27%, respectively, while the diffusion coefficient of acetonitrile did not change. The chemical shift and Nuclear Overhauser Effect Spectroscopy data confirmed formation of hydrogen bonds between the carboxyl group of I and residual water at low concentrations of I in acetonitrile, while IR-Raman data suggest formation of weak hydrogen bonds between I and acetonitrile at higher concentrations. In contrast, the diffusion coefficients of I and ethanol decreased by up to 15% as the concentration of I in ethanol was increased from 3.6 × 10–4 to 0.3 M. This together with IR and Raman data suggest formation of strong intermolecular hydrogen bonds between I and ethanol. In summary, our data suggest that solute–solvent interactions determine the critical supersaturation at the onset of nucleation (σcr) and thereby control the preferential crystallization of I polymorphs from supersaturated solutions of acetonitrile and ethanol. Furthermore, our data highlight the importance of intermolecular interactions between solute and residual water in aprotic solvents, such as acetonitrile.

Macromolecular Recognition: Interaction of Cyclodextrins with an Alternating Copolymer of Sodium Maleate and Dodecyl Vinyl Ether

AbstractInteraction of cyclodextrins (CDs) with an alternating copolymer of sodium maleate and dodecyl vinyl ether (pC12MA), which forms micelle‐like aggregates in aqueous media, is investigated by several NMR techniques to explore the competition of the complexation of CDs with the dodecyl (C12) groups and the self‐association of the C12 groups. 1H NMR and two‐dimensional nuclear Overhauser effect spectroscopy data demonstrate that α‐CD interacts significantly with the C12 groups in pC12MA but β‐ or γ‐CD does not, which indicates that the competition with self‐association of the C12 groups enhances the selectivity towards α‐CD. Furthermore, the binding isotherm prepared using 1H NMR data exhibits a sigmoidal curve, which is indicative of cooperative complexation of α‐CD with pC12MA.magnified image

A topology‐constrained distance network algorithm for protein structure determination from NOESY data

This article formulates the multidimensional nuclear Overhauser effect spectroscopy (NOESY) interpretation problem using graph theory and presents a novel, bottom-up, topology-constrained distance network analysis algorithm for NOESY cross peak interpretation using assigned resonances. AutoStructure is a software suite that implements this topology-constrained distance network analysis algorithm and iteratively generates structures using the three-dimensional (3D) protein structure calculation programs XPLOR/CNS or DYANA. The minimum input for AutoStructure includes the amino acid sequence, a list of resonance assignments, and lists of 2D, 3D, and/or 4D-NOESY cross peaks. AutoStructure can also analyze homodimeric proteins when X-filtered NOESY experiments are available. The quality of input data and final 3D structures is evaluated using recall, precision, and F-measure (RPF) scores, a statistical measure of goodness of fit with the input data. AutoStructure has been tested on three protein NMR data sets for which high-quality structures have previously been solved by an expert, and yields comparable high-quality distance constraint lists and 3D protein structures in hours. We also compare several protein structures determined using AutoStructure with corresponding homologous proteins determined with other independent methods. The program has been used in more than two dozen protein structure determinations, several of which have already been published.

Solution structure of two xenoantigens: alpha Gal-LacNAc and alpha Gal-Lewis X.

Organ hyperacute rejection, a phenomenon occurring during discordant xenotransplantation, is due to the recognition of an oligosaccharide epitope by human xenoreactive natural antibodies. In addition to the alpha Gal(1-3)beta Gal(1-4)GlcNAc trisaccharide, a fucosylated structure, alpha Gal-Lewis X, has been shown to be recognized by the antibodies. Both the trisaccharide and the tetrasaccharide have been synthesized by chemical methods. A complete nuclear magnetic resonance characterization of the two compounds has been performed, including the measurements of two-dimensional nuclear Overhauser effect spectroscopy data. Molecular dynamics simulations were run for several ns in the presence of explicit water molecules. The combination of experimental and theoretical approaches revealed the effect of an additional fucose residue on the conformational behavior of the xenoantigen. This branched fucose strongly rigidifies the N-acetyllactosamine. The effect on the alpha Gal(1-3)Gal fragment is less marked. In the presence of fucose, the terminal alpha Gal residue can still adopt two different conformations, but the equilibrium populations are modified.

A flexible model for the cell wall polysaccharide of Streptococcus mitis J22 determined by three-dimensional 13C edited nuclear overhauser effect spectroscopy and 13C-1H long-range coupling constants combined with molecular modeling.

We report on the conformation of a tetrasaccharide fragment in the repeating subunit of the cell wall polysaccharide of Streptococcus mitis J22, a receptor for the lectin of Actinomyces viscosus T14V in a bacterial coaggregation that is important in the ecological interactions of oral bacteria. Although there is considerable overlap of the 1H-nmr signals, some cross peaks can be extracted from conventional two-dimensional nuclear Overhauser effect spectroscopy (NOESY) data on the polysaccharide. These data cannot be fit to a single conformation of the tetrasaccharide fragment. Therefore we have prepared a polysaccharide sample fully enriched in 13C from which we have determined accurate NOESY cross-peak volumes in a three-dimensional heteronuclear-resolved spectrum that allows accurate determination of many more NOESY cross peaks than does conventional two-dimensional spectroscopy. We have also used the 13C enriched polysaccharide to measure accurate values of long-range 13C-1H coupling constants that can be correlated with glycosidic dihedral angles. Molecular modeling calculations on the polysaccharide fragment, including molecular dynamics simulations, identify multiple low-energy conformations. This result is to be contrasted with previous calculations on blood group oligosaccharides in our laboratory using similar methods that showed relatively rigid conformations with little flexibility of the glycosidic linkages. The present NOESY and 3JCH data can be reconciled with a model for the antigenic tetrasaccharide in which three distinct conformations are in fast exchange. We propose that some carbohydrate epitopes such as those of the blood group oligosaccharides are relatively rigid while others such as the tetrasaccharide fragment in these studies exhibit much greater flexibility.

A flexible model for the cell wall polysaccharide of Streptococcus mitis J22 determined by three-dimensional 13C edited nuclear overhauser effect spectroscopy and 13C-1H long-range coupling constants combined with molecular modeling

We report on the conformation of a tetrasaccharide fragment in the repeating subunit of the cell wall polysaccharide of Streptococcus mitis J22, a receptor for the lectin of Actinomyces viscosus T14V in a bacterial coaggregation that is important in the ecological interactions of oral bacteria. Although there is considerable overlap of the 1H-nmr signals, some cross peaks can be extracted from conventional two-dimensional nuclear Overhauser effect spectroscopy (NOESY) data on the polysaccharide. These data cannot be fit to a single conformation of the tetrasaccharide fragment. Therefore we have prepared a polysaccharide sample fully enriched in 13C from which we have determined accurate NOESY cross-peak volumes in a three-dimensional heteronuclear-resolved spectrum that allows accurate determination of many more NOESY cross peaks than does conventional two-dimensional spectroscopy. We have also used the 13C enriched polysaccharide to measure accurate values of long-range 13C-1H coupling constants that can be correlated with glycosidic dihedral angles. Molecular modeling calculations on the polysaccharide fragment, including molecular dynamics simulations, identify multiple low-energy conformations. This result is to be contrasted with previous calculations on blood group oligosaccharides in our laboratory using similar methods that showed relatively rigid conformations with little flexibility of the glycosidic linkages. The present NOESY and 3JCH data can be reconciled with a model for the antigenic tetrasaccharide in which three distinct conformations are in fast exchange. We propose that some carbohydrate epitopes such as those of the blood group oligosaccharides are relatively rigid while others such as the tetrasaccharide fragment in these studies exhibit much greater flexibility. © 1996 John Wiley & Sons, Inc.

Conformational requirements of suramin to target angiogenic growth factors.

The conformational requirements of suramin for its binding to basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) were examined by molecular modeling and docking simulations using the conformational features of suramin determined by the present proton nuclear magnetic resonance (1H-NMR) studies and the crystal structures of growth factors reported previously. The assignment of resonances of suramin to individual protons was accomplished by the combined analysis of the coupling constants, two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY). The NOESY data obtained for suramin were utilized in a conformational search algorithm with constraints to generate a family of conformers which were further refined by restrained energy minimization. A family of nine conformers generated by restrained modeling falls primarily into one of two categories, either the conformer's two naphthyl rings are far apart, approximately 28-30 A, from one another or the conformer's two naphthyl rings are relatively close, approximately 16-18 A. The NMR data provide evidence for the presence of more than one conformer in solution. The modeling and docking simulation studies suggest that suramin binds efficiently to bFGF and PDGF by an induced-fit mechanism, wherein suramin complements bFGF or PDGF by adjusting its conformational freedom around the two pairs of single bonds that link the middle phenyl rings to the secondary amide backbone. The interaction of suramin with bFGF or PDGF primarily involves ion-pair, hydrophobic and hydrogen bonding interactions, in addition to van der Waals' contacts. The results indicate that suramin not only sterically blocks the receptor binding loop of the growth factors, but also competes for the binding sites of agonists such as heparin. The results suggest that suramin's propensity to bind to several polypeptides of varying size and structure is due to its conformational flexibility. Collectively, the data emphasize that conformationally constrained suramin analogs that selectively and competitively target angiogenic growth factors could be designed to reduce non-specific binding and, accordingly, toxicity.

Conformational mimicry: synthesis and solution conformation of a cyclic somatostatin hexapeptide containing a tetrazole cis amide bond surrogate.

Potent, cyclic hexapeptide analogues of somatostatin are generally believed to adopt some common secondary structural features: a II' beta turn at one end of the cycle, and a type VI turn with a cis amide bond at the other. A proposed cis amide surrogate, the 1,5-disubstituted tetrazole, has been placed into a cyclic hexapeptide analog of somatostatin in order to constrain the putative cis amide bond. The final cyclization was done by either chemical or enzymatic means. The product, cyclo(Ala6-Tyr7-D-Trp8-Lys9-Val10-Phe11-psi[CN4] ), was found to have 83% of the activity of somatostatin. Solution nmr analysis in DMSO/water revealed that the backbone as well as side chain chi1 and chi2 were well ordered. Relaxation matrix methods were used to extract distance restraints from the nuclear Overhauser effect spectroscopy data set, and these were used in a systematic search of torsional space to identify structures consistent with the nmr data. Restrained minimizations of these structures using a number of different force fields produced structures having the expected beta II' turn at D-Trp8-Lys9 and a beta VIa turn in the Phe11-psi[CN4]-Ala6 portion of the molecule. The similarity of the minimized structures to those previously reported for cyclic hexapeptide analogues of somatostatin confirms the similarity of the tetrazole geometry to that of the cis amide in solution.

Solution structure of the variant-3 neurotoxin from Centruroides sculpturatus Ewing.

The solution structure of the CsE-v3 neurotoxin from the venom of the North American scorpion Centruroides sculpturatus Ewing (CsE) has been determined by a hybrid refinement procedure that employed distance geometry and dynamical simulated annealing. Distance constraints deduced from the nuclear Overhauser effect spectroscopy data and torsion angle constraints deduced from the vicinal coupling constant data were used in the refinement procedure. A family of simulated annealing structures that showed no constraint violations was generated. The energy-minimized average structure exhibited root-mean-square deviations of 0.121 nm for the backbone and 0.182 nm for all atoms, with respect to this family. These studies confirm the previously qualitative NMR findings about the secondary structural features, viz. the presence of a short alpha-helix composed of residues 23-31 and an antiparallel beta-sheet composed of the strands of residues 1-5, 45-50 and 36-42. A cluster of aromatic ring systems is located on one side of the protein. The solution and crystal structures have similar overall features, but show some minor differences.

Конформационный анализ фрагмента 1–45 аденилаткиназы кролика на основе данных двухмерной спектроскопии ядерного эффекта Оверхаузера

Конформационный анализ фрагмента 1–45 аденилаткиназы кролика на основе данных двухмерной спектроскопии ядерного эффекта Оверхаузера