The Flexibility of Oligosaccharides Unveiled Through Residual Dipolar Coupling Analysis

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments.

Bimodal 1H Double Quantum Build-Up Curves by Fourier and Laplace-like Transforms on Aged Cross-Linked Natural Rubber

The 1H DQ Fourier and Laplace-like spectra for a series of cross-linked natural rubber (NR) samples naturally aged during six years are presented and characterized. The DQ build-up curves of these samples present two peaks which cannot be described by classical functions. The DQ Fourier spectra can be obtained after a numeric procedure which introduces a correction time which depends less on the chosen approximation, spin-½ and isolated CH2 and CH3 functional groups. The DQ Fourier spectra are well described by the distributions of the residual dipolar coupling correlated with the distribution of the end-to-end vector of the polymer network, and with the second and fourth van Vleck moments. The deconvolution of DQ Fourier spectra with a sum of four Gaussian variates show that the center and the width of Gaussian functions increase linearly with the increase in the cross-link density. The Laplace-like spectra for the natural aged NR DQ build-up curves are presented. The centers of four Gaussian distributions obtained via both methods are consistent. The differences between the Fourier and Laplace-like spectra consist mainly of the spectral resolution in the favor of Laplace-like spectra. The last one was used to discuss the effect of natural aging for cross-linked NR.

Water-Soluble Glycosylated Resveratrol for Measuring Residual Dipolar Coupling in NMR

Residual dipolar coupling (RDC) is a newly emerging technique for obtaining more precise conformational features of molecules in nuclear magnetic resonance (NMR), which can be observed when the molecules are oriented in NMR sample solutions. Unlike the case of solubilization of resveratrol (Res) in aqueous media by surfactants, multiglycosylated resveratrol (GlyRes) was dispersed in aqueous media forming nanoparticles with an extremely small size. In the presence of an NMR alignment medium of bacteriophage, GlyRes molecules were efficiently oriented in the strong magnetic field allowing for observations of RDCs in NMR. Structural refinement using the observed RDC values enabled differentiation of trans and cis forms of Res.

2-amino-1,3-benzothiazole-6-carboxamide Preferentially Binds the Tandem Mismatch Motif r(UY:GA)

RNA helices are often punctuated with non-Watson-Crick features that can be the target of chemical compounds, but progress towards identifying small molecules specific for non-canonical elements has been slow. We have used a tandem UU:GA mismatch motif (5’-UG-3’:5’-AU-3’) embedded within the helix of an RNA hairpin as a model to identify compounds that bind the motif specifically. The three-dimensional structure of the RNA hairpin and its interaction with a small molecule compound identified through a virtual screen are presented. The G-A of the mismatch forms a sheared pair upon which the U-U base pair stacks. The hydrogen bond configuration of the U-U pair involves the O2 of the U adjacent to the G and the O4 of the U adjacent to the A. The G-A and U-U pairs are flanked by A-U and G-C base pairs, respectively, and the mismatch exhibits greater stability than when the motif is within the context of other flanking base pairs or when the 5’-3’ orientation of the G-A and U-U is swapped. Residual dipolar coupling constants were used to generate an ensemble of structures against which a virtual screen of 64,480 small molecules was performed to identify candidate compounds that the motif specifically binds. The tandem mismatch was found to be specific for one compound, 2-amino-1,3-benzothiazole-6-carboxamide, which binds with moderate affinity but extends the motif to include the flanking A-U and G-C base pairs. The finding that affinity for the UU:GA mismatch is flanking sequence dependent emphasizes the importance of motif context and potentially increases the number of small non-canonical features within RNA that can be specifically targeted by small molecules.

Protein structure prediction using sparse NOE and RDC restraints with Rosetta in CASP13

Computational methods that produce accurate protein structure models from limited experimental data, e.g. from nuclear magnetic resonance (NMR) spectroscopy, hold great potential for biomedical research. The NMR-assisted modeling challenge in CASP13 provided a blind test to explore the capabilities and limitations of current modeling techniques in leveraging NMR data which had high sparsity, ambiguity and error rate for protein structure prediction. We describe our approach to predict the structure of these proteins leveraging the Rosetta software suite. Protein structure models were predictedde novousing a two-stage protocol. First, low-resolution models were generated with the Rosettade novomethod guided by non-ambiguous nuclear Overhauser effect (NOE) contacts and residual dipolar coupling (RDC) restraints. Second, iterative model hybridization and fragment insertion with the Rosetta comparative modeling method was used to refine and regularize models guided by all ambiguous and non-ambiguous NOE contacts and RDCs. Nine out of 16 of the Rosettade novomodels had the correct fold (GDT-TS score >45) and in three cases high-resolution models were achieved (RMSD <3.5 Å). We also show that a meta-approach applying iterative Rosetta+NMR refinement on server-predicted models which employed non-NMR-contacts and structural templates leads to substantial improvement in model quality. Integrating these data-assisted refinement strategies with innovative non-data-assisted approaches which became possible in CASP13 such as high precision contact prediction will in the near future enable structure determination for large proteins that are outside of the realm of conventional NMR.