15 research outputs found
Tracking Alignment from the Moment of Inertia Tensor (TRAMITE) of Biomolecules in Neutral Dilute Liquid Crystal Solutions
Mutational, NMR, and NH Exchange Studies of the Tight and Selective Binding of 8-Oxo-dGMP by the MutT Pyrophosphohydrolase â
Solution Structure and NH Exchange Studies of the MutT Pyrophosphohydrolase Complexed with Mg 2+
Uncovering Nonconventional and Conventional Hydrogen Bonds in Oligosaccharides through NMR Experiments and Molecular Modeling: Application to Sialyl Lewis-X
Half-of-the-Sites Binding of Reactive Intermediates and Their Analogues to 4-Oxalocrotonate Tautomerase and Induced Structural Asymmetry of the Enzyme â
Glycan OH Exchange Rate Determination in Aqueous Solution: Seeking Evidence for Transient Hydrogen Bonds
Hydrogen bonds (Hbonds)
are important stabilizing forces in biomolecules.
However, for glycans in aqueous solution, direct NMR detection of
Hbonds is elusive because of their transient nature. Here, we present
Isotope-based Natural-abundance TOtal correlation eXchange SpectroscopY
(INTOXSY), a new <sup>1</sup>Hâ<sup>13</sup>C heteronuclear
single quantum coherenceâtotal correlation spectroscopy based
method, to extract OH groupsâ exchange rate constants (<i>k</i><sub>ex</sub>) for molecules in natural <sup>13</sup>C
abundance and show that OH Hbonds can be inferred from âslowerâ
H/D <i>k</i><sub>ex</sub>. We evaluate <i>k</i><sub>ex</sub> measured with INTOXSY in light of those extracted with
line-shape analysis. Subsequently, we use a set of common glycans
to establish a <i>k</i><sub>ex</sub> reference basis set
and to infer the existence of transient Hbonds involving OH donor
groups. Then, we report <i>k</i><sub>ex</sub> values for
a series of mono- and disaccharides, as well as for oligosaccharides
sialyl Lewis X and ÎČ-cyclodextrin, and compare the results with
those from the reference set to extract Hbond information. Finally,
we utilize NMR experimental data in conjunction with molecular dynamics
simulations to establish donor and acceptor Hbond pairs. Our exchange
rate measurements indicate that OH/OD exchange rates, <i>k</i><sub>HD</sub>, values <10 s<sup>â1</sup> are consistent
with transient Hbond OH groups and potential acceptor groups can be
uncovered through MD simulations
Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding
Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn-to-Ala substitutions at the predicted N-glycosylation sites of the M41-RBD were evaluated along with two control Val-to-Ala substitutions. CD analysis revealed that the secondary structure of all variants was retained compared with the unmodified M41-RBD construct. Six of the ten glycosylation variants lost binding to chicken trachea tissue and an ELISA-presented α2,3-linked sialic acid oligosaccharide ligand. LC/MSE glycomics analysis revealed that glycosylation sites have specific proportions of N-glycan subtypes. Overall glycosylation patterns of most variant RBDs were highly similar to those of the unmodified M41-RBD construct. In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. Combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the M41 spike protein
Uncovering Nonconventional and Conventional Hydrogen Bonds in Oligosaccharides through NMR Experiments and Molecular Modeling: Application to Sialyl LewisâX
We
describe the direct NMR detection of a CâH···O
nonconventional hydrogen bond (Hbond) and provide experimental and
theoretical evidence for conventional Hbonds in the pentasaccharide
sialyl Lewis-X (sLe<sup>X</sup>-5) between 5 and 37 °C in water.
Extensive NMR structural studies together with molecular dynamics
simulations offer strong evidence for significant local dynamics in
the Le<sup>X</sup> core and for previously undetected conventional
Hbonds in rapid equilibrium that modulate structure. These NMR studies
also showed temperature-dependent <sup>1</sup>H and <sup>13</sup>C
line broadening. The resulting model emerging from this study is more
complex than a simple rigid core description of Le<sup>X</sup>-like
molecules and improves our understanding of stabilizing interactions
in glycans
The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase:A kinetic, NMR, and mutational analysis
trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R(G)) disappeared in the spectrum of the inactive alphaR8A mutant (deltaH = 7.48 ppm, deltaN = 89.6 ppm), thereby assigning these resonances to alphaArg-11NepsilonH, and alphaArg-8NepsilonH, respectively. (1)H-(15)N-HSQC titration of the enzyme with the substrate analogue 3-chloro-2-butenoic acid (3-CBA), a competitive inhibitor (K(I)(slope) = 0.35 +/- 0.06 mM), resulted in progressive downfield shifts of the alphaArg-8Nepsilon resonance yielding a K(D) = 0.77 +/- 0.44 mM, comparable to the (K(I)(slope), suggestive of active site binding. Increasing the pH of free CaaD to 8.9 at 5 degrees C resulted in the disappearance of all nine Arg-NepsilonH resonances due to base-catalyzed NepsilonH exchange. Saturating the enzyme with 3-CBA (16 mM) induced the reappearance of two NepsilonH signals, those of alphaArg-8 and alphaArg-11, indicating that the binding of the substrate analogue 3-CBA selectively slows the NepsilonH exchange rates of these two arginine residues. The kinetic and NMR data thus indicate that betaPro-1 is the acid catalyst, alphaGlu-52 is a reasonable candidate for the general base, and alphaArg-8 and alphaArg-11 participate in substrate binding and in stabilizing the aci-carboxylate intermediate in a Michael addition mechanism