Spin Coupling in a Lewis Trisaccharide as Evidence of Inter-Residue C-H···O Hydrogen Bonding in Aqueous Solution.

Prior studies of the solution conformation of the Lewis (Le) trisaccharide, αFuc-(1→3)[βGal-(1→4)]-βGlcNAc, suggest that nonclassical inter-residue C-H···O hydrogen bonding in aqueous solution contributes to the stabilization of its 3D structure and affects its biological properties. Experimental evidence for this hydrogen bond in aqueous solution has been reported in the form of a NMR spin-coupling constant between C5'Fuc and H1″Gal measured by 2D NMR methods in unlabeled samples. A methyl glycoside of Le (MeβLe) was prepared containing selective C-labeling at C5'Fuc, and the H1″Gal signal was examined in high-field H NMR spectra for evidence of splitting or line-broadening caused by the C at C5'Fuc.

Context Effects on Human Milk Oligosaccharide Linkage Conformation and Dynamics Revealed by Analysis.

An emerging NMR method, analysis, has been applied to investigate context effects on the conformational properties of several human milk oligosaccharides (HMOs). The model of the β-(1→4) linkage in the disaccharide, methyl β-lactoside (MeL), was compared to those obtained for the same linkage in the HMO trisaccharides, methyl 2'-fucosyllactoside (Me2'FL) and methyl 3-fucosyllactoside (Me3FL), and in the tetrasaccharide, methyl 2',3-difucosyllactoside (Me2',3DFL). analysis revealed significant context effects on the mean values and circular standard deviations (CSDs) of the psi (ψ) torsion angles in these linkages.

analysis of the -glycosidic linkages of oligosaccharides using nonconventional NMR -couplings: and MD models of .

analysis (Meredith , 2022, , 3135-3141) is a new NMR-based method to treat ensembles of redundant NMR spin-coupling constants (-couplings) to obtain experiment-based probability distributions of molecular torsion angles in solution. Work reported to date on modeling the conformations of -glycosidic linkages of oligosaccharides using three conventional -coupling constraints ( , , ) has shown that the method gives mean torsion angles and circular standard deviations (CSDs) for in very good agreement with those obtained by MD simulation. On the other hand, CSDs for determined by analysis have consistently been much larger than those determined by MD, calling into question either the reliability of analysis or MD to accurately predict this behavior.

Conformational disorder in the crystal structure of methyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (methyl β-chitobioside) methanol monosolvate.

Methyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (methyl β-chitobioside), (IV), crystallizes from aqueous methanol at room temperature to give a structure (CHNO·CHOH) containing conformational disorder in the exocyclic hydroxymethyl group of one of its βGlcNAc residues. As observed in other X-ray structures of disaccharides containing β-(1→4) O-glycosidic linkages, inter-residue hydrogen bonding between O3H of the βGlcNAc bearing the OCH aglycone and O5 of the adjacent βGlcNAc is observed based on the 2.79 Å internuclear distance between the O atoms.

Analysis: Unbiased Multi-State Conformational Modeling of Exocyclic Hydroxymethyl Group Conformation in Methyl Aldohexopyranosides.

analysis (. , , 3135-3141) has been applied to model exocyclic hydroxymethyl group conformation in methyl β-D-glucopyranoside (βGlcOMe), methyl β-D-galactopyranoside (βGalOMe), and methyl β-D-mannopyranoside (βManOMe) in an unbiased manner. Using up to eight NMR -couplings sensitive to rotation about the C5-C6 bond (torsion angle ω), two-state models of ω were obtained that are qualitatively consistent with the relative populations of the , and rotamers reported previously.

Methyl α-D-galactopyranosyl-(1→3)-β-D-galactopyranoside and methyl β-D-galactopyranosyl-(1→3)-β-D-galactopyranoside: Glycosidic linkage conformation determined from MA'AT analysis.

MA'AT analysis has been applied to two biologically-important O-glycosidic linkages in two disaccharides, α-D-Galp-(1→3)-β-D-GalpOMe (3) and β-D-Galp-(1→3)-β-D-GalpOMe (4). Using density functional theory (DFT) to obtain parameterized equations relating a group of trans-O-glycosidic NMR spin-couplings to either phi (ϕ') or psi (ψ'), and experimental J, J, and J spin-couplings measured in aqueous solution in C-labeled isotopomers, probability distributions of ϕ' and ψ' in each linkage were determined and compared to those determined by aqueous 1-μs molecular dynamics (MD) simulation. Good agreement was found between the MA'AT and single-state MD conformational models of these linkages for the most part, with modest (approximately <15°) differences in the mean values of ϕ' and ψ', although the envelope of allowed angles (encoded in circular standard deviations or CSDs) is consistently larger for ϕ' determined from MA'AT analysis than from MD for both linkages.

One-bond C-C spin-coupling constants in saccharides: a comparison of experimental and calculated values by density functional theory using solid-state C NMR and X-ray crystallography.

Methyl aldohexopyranosides were C-labeled at contiguous carbons, crystallized, and studied by single-crystal X-ray crystallography and solid-state C nuclear magnetic resonance (NMR) spectroscopy to examine the degree to which density functional theory (DFT) can calculate one-bond C-C spin-coupling constants () in saccharides with sufficient accuracy to permit their use in ' analysis, a newly-reported hybrid DFT/NMR method that provides probability distributions of molecular torsion angles in solution (Zhang , , 2017, , 3042-3058; Meredith , , 2022, , 3135-3141). Experimental values in crystalline samples of the doubly C-labeled compounds were measured by solid-state C NMR and compared to those calculated from five different DFT models: (1) values calculated from single structures identical to those observed in crystalline samples by X-ray crystallography (all atom refinement); (2) values calculated from the single structures in (1) but after Hirshfeld atom refinement (HAR); (3) values calculated from the single structures in (1) after DFT-optimization of hydrogen atoms only; and (4 and 5) values calculated in rotamers of torsion angle (C1-C2-O2-O2H) or (C4-C5-C6-O6) from which either specific or generalized parameterized equations were obtained and used to calculate values in the specific or rotamers observed in crystalline samples. Good qualitative agreement was observed between calculated values and those measured by solid-state C NMR regardless of the DFT model, but in no cases were calculated values quantitative, differing (over-estimated) on average by 4-5% from experimental values.

-Acetyl Side-Chain Conformation in Saccharides: Solution Models Obtained from Analysis.

analysis has been applied to model the conformational properties of -acetyl side-chains in biologically important GlcNAc and ManNAc monosaccharides and in a βGlcNAc-(1→4)-βGlcNAc disaccharide. Density functional theory calculations were conducted to obtain parameterized equations that relate the magnitudes and signs of 10 spin-coupling constants to conformations of the C2-N2 bonds of GlcNAc and ManNAc. Six of these equations were used with experimental -couplings, measured in HO/HO and DMSO- solvents in selectively C-labeled compounds, to model the C1-C2-N2-C1' torsion angle (θ) in GlcNAc and ManNAc residues.

C-C spin-coupling constants in crystalline C-labeled saccharides: conformational effects interrogated by solid-state C NMR spectroscopy.

Solid-state C NMR spectroscopy has been used in conjunction with selectively C-labeled mono- and disaccharides to measure C-C spin-couplings (J) in crystalline samples. This experimental approach allows direct correlation of J values with specific molecular conformations since, in crystalline samples, molecular conformation is essentially static and can be determined by X-ray crystallography. J values measured in the solid-state in known molecular conformations can then be compared to corresponding J values calculated in the same conformations using density functional theory (DFT).

Synthesis and O-Glycosidic Linkage Conformational Analysis of C-Labeled Oligosaccharide Fragments of an Antifreeze Glycolipid.

NMR studies of two C-labeled disaccharides and a tetrasaccharide were undertaken that comprise the backbone of a novel thermal hysteresis glycolipid containing a linear glycan sequence of alternating [βXyl p-(1→4)-βMan p-(1→4)] dimers. Experimental trans-glycoside NMR J-couplings, parameterized equations obtained from density functional theory (DFT) calculations, and an in-house circular statistics package ( MA'AT) were used to derive conformational models of linkage torsion angles ϕ and ψ in solution, which were compared to those obtained from molecular dynamics simulations. Modeling using different probability distribution functions showed that MA'AT models of ϕ in βMan(1→4)βXyl and βXyl(1→4)βMan linkages are very similar in the disaccharide building blocks, whereas MA'AT models of ψ differ.

Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-X.

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-X or BCL2L1).

System effectiveness of detection, brief intervention and refer to treatment for the people with post-traumatic emotional distress by MERS: a case report of community-based proactive intervention in South Korea.

Background: Korea has experienced diverse kind of disasters these days. Among them the 2015 middle eastern respiratory syndrome (MERS) outbreak imposed great psychological stress on almost all Korean citizens. Following the MERS outbreak, government is reviewing overall infectious disease management system and prioritizing the establishment of mental health service systems for infectious disease.

The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases.

The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21(Cip1), p27(Kip1) and p57(Kip2). Their kinase inhibitory activities are mediated by a homologous N-terminal kinase inhibitory domain. The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the kinase inhibitory domain and a threonine residue within the flexible C-terminus.

Structure and apoptotic function of p73.

p73 is a structural and functional homologue of the p53 tumor suppressor protein. Like p53, p73 induces apoptosis and cell cycle arrest and transactivates p53-responsive genes, conferring its tumor suppressive activity. In addition, p73 has unique roles in neuronal development and differentiation.

Gender-specific metabolomic profiling of obesity in leptin-deficient ob/ob mice by 1H NMR spectroscopy.

Despite the numerous metabolic studies on obesity, gender bias in obesity has rarely been investigated. Here, we report the metabolomic analysis of obesity by using leptin-deficient ob/ob mice based on the gender. Metabolomic analyses of urine and serum from ob/ob mice compared with those from C57BL/6J lean mice, based on the (1)H NMR spectroscopy in combination with multivariate statistical analysis, revealed clear metabolic differences between obese and lean mice.

Dual-site interactions of p53 protein transactivation domain with anti-apoptotic Bcl-2 family proteins reveal a highly convergent mechanism of divergent p53 pathways.

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins.

Disorder-function relationships for the cell cycle regulatory proteins p21 and p27.

The classic structure-function paradigm has been challenged by a recently identified class of proteins: intrinsically disordered proteins (IDPs). Despite their lack of stable secondary or tertiary structure, IDPs are prevalent in all forms of life and perform myriad cellular functions, including signaling and regulation. Importantly, disruption of IDP homeostasis is associated with numerous human diseases, including cancer and neurodegeneration.

Cell signaling, post-translational protein modifications and NMR spectroscopy.

Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy.

Cell cycle regulation by the intrinsically disordered proteins p21 and p27.

Today, it is widely accepted that proteins that lack highly defined globular three-dimensional structures, termed IDPs (intrinsically disordered proteins), play key roles in myriad biological processes. Our understanding of how intrinsic disorder mediates biological function is, however, incomplete. In the present paper, we review disorder-mediated cell cycle regulation by two intrinsically disordered proteins, p21 and p27.

Residual structure within the disordered C-terminal segment of p21(Waf1/Cip1/Sdi1) and its implications for molecular recognition.

Probably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein-protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets.

Structural basis for the conformational integrity of the Arabidopsis thaliana HY5 leucine zipper homodimer.

The leucine zipper (LZ) domain of the HY5 transcription factor from Arabidopsis thaliana has unique primary structural properties, including major occupation by the Leu residues as well as two buried polar residues in the a positions and a localized distribution of charged and polar residues in the first three heptad repeats. In this study, we solved the crystal structure of the HY5 LZ domain and show that the peculiarities in the primary sequence yield unusual structural characteristics. For example, the HY5 LZ domain exhibits a bipartite charge distribution characterized by a highly negative electrostatic surface potential in its N-terminal half and a nearly neutral potential in its C-terminal half.

Intrinsically unstructured N-terminal domain of bZIP transcription factor HY5.

The Arabidopsis HY5 protein is a basic leucine zipper (bZIP) transcription factor that promotes photomorphogenesis. HY5 binds directly to the promoters of light responsible element containing the G-box and thus regulates their transcriptional activity. The level and activity of HY5 are negatively regulated, in a light-dependent manner, by interaction with the COP1 protein, which targets HY5 for proteasome-mediated degradation in the nucleus.