Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derived peptide with vasoconstricting activity, was determined in an aqueous solution by means of a combination of NMR and distance geometry calculations. The resulting structure is characterized by an alpha-helical conformation in the sequence region, Lys9-Cys15. Furthermore, an extended structure and a turn structure exist in the Cys1-Ser4 and Ser5-Asp8 regions respectively, and no preferred conformation was found for the C-terminal part of the peptide which was not uniquely constrained by the NMR data. These structural elements, the alpha-helical structure in the sequence portion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys, are homologous to those found commonly in several neurotoxic peptides.     

The solution conformations of ferrichrome and deferriferrichrome determined by 1H-NMR spectroscopy and computational modeling

We have applied computational procedures that utilize nmr data to model the solution conformation of ferrichrome, a rigid microbial iron transport cyclohexapeptide of known x-ray crystallographic structure [D. van der Helm et al. (1980) J. Am. Chem. Soc. 102, 4224-4231]. The Al3+ and Ga3+ diamagnetic analogues, alumichrome and gallichrome, dissolved in d6-dimethylsulfoxide (d6-DMSO), were investigated via one- and two-dimensional 1H-nmr spectroscopy at 300, 600, and 620 MHz. Interproton distance constraints derived from proton Overhauser experiments were input to a distance geometry algorithm [T. F. Havel and K. Wüthrich (1984) Bull. Math. Biol. 46, 673-691] in order to generate a family of ferrichrome structures consistent with the experimental data. These models were subsequently optimized through restrained molecular dynamics/energy minimization [B. R. Brooks et al. (1983) J. Comp. Chem. 4, 187-217]. The resulting structures were characterized in terms of relative energies and conformational properties. Computations based on integration of the generalized Bloch equations for the complete molecule, which include the 14N-1H dipolar interaction, demonstrate that the x-ray coordinates reproduce the experimental nuclear Overhauser effect time courses very well, and indicate that there are no significant differences between the crystalline and solution conformations of ferrichrome. A similar study of the metal free peptide, deferriferrichrome, suggests that at least two conformers are present in d6-DMSO at 23 degrees C. Both are different from the ferrichrome structure and explain, through conformational averaging, the observed amide NH and CH alpha multiplet splittings. The occurrence of interconverting peptide backbone conformations yields an increased number of sequential NH-CH alpha and NH-NH Overhauser connectivities, which reflects the mean value of r-6 dependence of the dipolar interaction. Our results support the idea that, in the case of structurally rigid peptides, moderately accurate distance constraints define a conformational subspace encompassing the "true" structure, and that energy considerations reduce the size of this subspace. For flexible peptides, however, the straight-forward approach can be misleading since the nmr parameters are averaged over substantially different conformational states.     

Solution structures of alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.     

Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry

Although deamidation at asparagine and glutamine has been found in numerous studies of a variety of proteins, in almost all cases the analytical methodology that was used could detect only a single site of deamidation. For the extensively studied case of reduced bovine ribonuclease A (13,689 Da), only Asn67 deamidation has been demonstrated previously, although one study found three monodeamidated fractions. Here top down tandem mass spectrometry shows that Asn67 deamidation is extensive before Asn71 and Asn94 react; these are more than half deamidated before Asn34 reacts, and its deamidation is extensive before that at Gln74 is initiated. Except for the initial Asn67 site, these large reactivity differences correlate poorly with neighboring amino acid identities and instead indicate residual conformational effects despite the strongly denaturing media that were used; deamidation at Asn67 could enhance that at Asn71, and these enhance that at Gln74. This success in the site-specific quantitation of deamidation in a 14 kDa protein mixture, despite the minimal 1 Da (-NH2 --> -OH) change in the molecular mass, is further evidence of the broad applicability of the top down MS/MS methodology for characterization of protein posttranslational modifications.     

Preferred conformations and dynamics of five core structures of mucin typeO-glycans determined by NMR spectroscopy and force field calculations

Glycosyltransferases acting onO-glycans have been shown to exhibit distinct specificity for the carbohydrate and the peptide moiety of their substrates. As an approach to study the 3-dimensional interactions between enzymes andO-glycan substrates, we determined the preferred conformations of five oligosaccharide-core structures of mucin type glycoproteins by NMR spectroscopy and by static and dynamic force field calculations. Seven oligosaccharides, representing five basic core structures, were investigated: Gal(1–3)GalNAcBzl (1, core 1), GlcNAc(1–6)[Gal(1–3)]GalNAcBzl (2, core 2), GlcNAc(1–3)GalNacBzl (3, core 3), GlcNAc(1–6)[GlcNAc(1–3)]GalNAcBzl (4, core 4), GlcNAc(1–6)GalNAcBzl (5, core 6), the elongated core 2, Gal(1–4)GlcNAc(1–6)[Gal(1–3)]GalNAcpNp (6) and GalNAc-Bzl (7). The dynamic behaviour of the molecules was studied by Metropolis Monte Carlo (MMC) simulations. Experimental coupling constants, chemical shift changes, and NOEs were compared with results from static energy minimizations and dynamic MMC simulations and show a good agreement. MMC simulations show that the (1–6) linkage is much more flexible than the (1–3) or the (1–4) linkages. The preferred conformations of the disaccharides (1) and (3) show only slight differences due to the additionalN-acetyl group in (3). The conformational equilibrium of (1–3) glycosidic bonds of1 and3 was not affected by attaching a (1–6) linked GlcNAc unit to the GalNAc residue in2 and4. However, experimental and theoretical data show that the (1–6) linkages of the trisaccharides2 and4, which carry an additional (1–3) linked glycosyl residue, change their preferred conformations when compared with (5). The 6-branch also shows significant interactions with the benzyl aglycon altering the preferred conformation of the hydroxymethyl group of the GalNAc to a higher proportion of the gt conformer. The (1–6) linkage of2, 4, and6 can have two different families of conformations of which the lower energy state is populated only to about 20% of the time whereas the other state with a relative enthalpy of 4 kcal mol^–1 is populated to 80%. This fact demonstrates that the two conformational states have different entropy contents. Entropy is implicitly included in MMC simulations but cannot be derived from energy minimizations.Abbreviations Bzl benzyl - COSY correlation spectroscopy - Gal d-galactose - GalNAc N-acetyl-d-galactosamine - GalNAc-ol N-acetylgalactosaminitol - GlcNAc N-acetyl-d-glucosamine - HOHAHA homonuclear Hartmann-Hahn-spectroscopy - MMC metropolis Monte Carlo - NOE nuclear Overhauser enhancement - pNp p-nitrophenyl - ROESY rotating frame Overhauser enhancement spectroscopy - TOCSY totally correlated spectroscopy     

Precise location of DNase I cutting sites in the nucleosome core determined by high resolution gel electrophoresis.

The precise locations of the DNase I cutting sites in the nucleosome core have been determined by analysis of the DNA products of a DNase I digestion of 32P end-labelled mucleosome cores on a high resolution gel electrophoresis system. This system is capable of resolving fragments of mixed sequence DNA differing by one base into the region of 160 bases in length. The DNase I cutting sites in the core are found to be spaced at multiples of about 10.4 (i.e. clearly different from 10.0) bases along the DNA, but show significant variations about this value. In addition to the location of the sites, the stagger between individual sites on opposite strands has been determined and is found to be inconsistent with at least one proposed mechanism for nuclease cleavage of chromatin DNA. Finally, a calculated distribution of fragment lengths in a DNase I digest of nuclei has been determined from the data obtained from the nucleosome core and found to be in reasonable agreement with the observed distribution. The periodicity of 10.4 is discussed with respect to the number of base pairs per turn of chromatin DNA and the number of superhelical turns of DNA per nucleosome.     

1H-NMR assignments of GM1-oligosaccharide in deuterated water at 500 MHz by two-dimensional spin-echo J-correlated spectroscopy

The ¹H-NMR spectra of the oligosaccharide derived from monosialoganglioside G_M1 (G_M1 = β-d-galactosyl-(1–3)-β-d-N-acetylgalactosaminyl-(1–4)-[α-N-acetylneuraminyl-(2–3)]-β-d-galactosyl-( 1–4)-β-d-glucosylceramide) (G_M1OS) and its reduced form (G_M1OS-R) have been obtained at 500 MHz in D₂O. Through the combined use of one-dimensional and homonuclear two-dimensional spin-echo J-correlated (2D SECSY) spectra of G_M1OS-R, the assignments for the ring protons of G_M1OS are made. Data on chemical shifts and coupling constants of G_M1OS including the α-linked neuraminic acid protons, in aqueous solution, are tabulated. Due to the very small coupling constants (<2 Hz) and the closeness in chemical shifts (<0.04 ppm) for the pair of correlated peaks in the two-dimensional spectrum, the information on the connectivities of the H5 ring protons of the neutral sugar residues is missing. Second-order coupling also blurs this information. Data are compared with those obtained for ganglioside G_M1 in dimethyl sulfoxide (DMSO;the actual composition therein was 97% DMSO-d₆ and 3% D₂O) by T.A.W. Koerner, J. H. Prestegard, P. C. Demou, and R. K. Yu (1983, Biochemistry22, 2676). While the heterogeneity of chemical shifts for the H5, H6a, and H6b protons diminishes in D₂O, that for A-9a and A-9b remains. The latter suggests an intraneuraminic acid conformation involving the glycerol side chain unaffected by the solvent. Moreover, the chemical shifts of the III-1, III-2, and A-4 protons (and perhaps the II-4, IV-2, and A-8 protons) in D₂O exhibit unusual upfield shifts compared with those in DMSO. This indicates that the intramolecular interactions between GalNAc residue III and neuraminic acid present in DMSO are weakened in D₂O. The effect of temperature on the conformation is also examined and appears to be minimal (<0.02 ppm) in the range 22–50 °C.     

Three-dimensional structure of (1-36)bacterioopsin in methanol-chloroform mixture and SDS micelles determined by 2D 1H-NMR spectroscopy.

Spatial structures of proteolytic segment A (sA) of bacterioopsin of H. halobium (residues 1-36) solubilized in a mixture of methanol-chloroform (1:1), 0.1 M LiClO4 organic mixture, or in perdeuterated sodium dodecyl sulfate (SDS) micelles, were determined by 2D 1H-NMR techniques. 324 and 400 NOESY cross-peak volumes were measured in NOESY spectra of sA in organic mixture and SDS micelles, respectively. The sA spatial structures were determined by local structure analysis, distance geometry calculation with program DIANA and systematic search for energetically allowed side chain rotamers consistent with NOESY cross-peak volumes. The structures of sA are similar in both milieus and have the right-handed alpha-helical region from Pro8 to Met32 with root mean square deviation (RMSD) of 0.25 A between backbone heavy atoms and fit well with Pro8 to Met32 alpha-helical region in electron cryo-microscopy model of bacteriorhodopsin. The N-terminal region Ala2-Gly6 of sA in organic mixture has a fixed structure of two consecutive gamma-turns as 2 * 2(7)-helix (RMSD of 0.25 A) stabilized by the Thr5 NH...O = C Gln3 and Ile4 NH...O = C Ala2 hydrogen bonds while this region in SDS micelles has disordered structure with RMSD of 1.44 A for backbone heavy atoms. The C-terminal region Gly33-Asp36 of sA is disordered in both milieus. Torsion angles chi 1 of sA were unequivocally determined for 13 (SDS) and 11 (organic mixture) of alpha-helical residues and are identical in both milieus.     

Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core.

We have isolated a nonfucosylated and three variously fucosylated neutral oligosaccharides from human milk that are based on the iso-lacto-N-octaose core. Their structures were characterized by the combined use of electrospray mass spectrometry (ES-MS) and NMR spectroscopy. The branching pattern and blood group-related Lewis determinants, together with partial sequences and linkages of these oligosaccharides, were initially elucidated by high-sensitivity ES-MS/MS analysis, and then their full structure assignment was completed by methylation analysis and 1H-NMR. Three new structures were identified. The nonfucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc, has not previously been reported as an individual oligosaccharide. The monofucosylated and trifucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc and Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6[Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3]Galbeta1-4Glc, both containing an internal Lex epitope, are also novel structures.     

Activation volumes of processes linked to the phototransformation of protochlorophyllide determined by fluorescence spectroscopy at high pressure

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 degrees C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol(-1), which corresponds with the formation of one H-bond/molecule.     

A study on human urine in a high-selenium area of China by 1H-NMR spectroscopy

In this study, high-resolution 600-MHz ¹H-NMR (nuclear magnetic resonance) spectroscopies were used to compare the urinary metabolic profiles of healthy humans and humans in a high-selenium area of China. NMR biomarkers for renal and liver lesions were observed by comparing the urine ¹H-NMR spectra. In urinary excretion, the concentrations in human urine samples of formate, lactate, acetate, hippurate, and alanine in overexposure to selenium were increased, whereas citrate, creatine, and TMAO excretion were decreased compared with that of the healthy human—some of them even disappeared. An interesting result was the appearance of formate in urine, which has previously been shown to lead to acidosis and chronic renal failure and interfere with the lumen and proximal tubular cells. The level of creatine was associated with the seminal activity. The changes of acetate and citrate may explain the disorder of the cellular energy metabolism caused by selenium, and the changes of other amino acids were a result of the reuptake of these compounds that had been blocked in the glomerulus and proximal tubule. The results elucidate the renal/liver lesion in humans in high-selenium area by ¹H-NMR spectroscopy and offer the molecular basic of selenium toxicity.     

1H-NMR spectroscopy on elongation factor Tu from Escherichia coli: Resolution enhancement by perdeuteration

¹H-NMRProteinEF-TuPerdeuteration     

C1 binding by murine IgM. The effect of a Pro-to-Ser exchange at residue 436 of the mu-chain

We have examined a defect in complement activation in a mutant trinitrophenyl-binding pentameric murine monoclonal IgM which has serine replacing the proline normally found at position 436 in the protein. The mutant protein showed equivalent hapten binding but a 100-fold decreased ability to initiate complement-dependent lysis of trinitrophenyl-coupled erythrocytes at physiological ionic strength (mu = 0.15). C4b deposition mediated by the mutant protein was impaired to a similar degree. C1 bound by the mutant protein showed C1s to C1-s conversion, suggesting normal activation. When measured at reduced ionic strength (mu = 0.06), the C1 and C1q binding affinity of the mutant protein was approximately one-half that of the wild type. However, the C1 binding affinity of the mutant protein showed a greater dependence upon ionic strength such that at physiological ionic strength we estimate a 50-fold lower C1 binding affinity for the mutant molecule. Kinetic studies suggested that this difference in affinity was largely attributable to differences in association rates. In addition, a fixed proportion of the mutant molecules showed no C1 binding. We conclude that the defect in complement activation occurs at the level of C1 binding. Our data support a role for the C mu 3 domain (residues 340-440) in C1 binding by IgM.     

Filamentous bacteriophage Pf1 structure determined at 7 Å resolution by refinement of models for the α-helical subunit

The molecular structure of filamentous bacteriophage Pf1 has been determined to 7 Å resolution by analysis of X-ray diffraction data from partially oriented fibers of virus particles. The continuous intensity distribution along layer-lines was measured by numerically separating contributions from overlapping layer-lines. The data were phased by an iterative refinement technique that used the known spatial extent and high α-helical content of the virus particle to refine a structural model. This refinement converges to a unique structural solution that is consistent with the X-ray data and with information derived from physical and chemical studies. The coat protein consists of two α-helical segments: one, almost parallel to the particle axis, is centered at a radius of about 15 Å; the other, at about 25 Å radius, is tilted by about 25 ° to the particle axis. This structure is consistent with every generalization about α-helical packing. The inner and outer segments interlock along most of their length with a crossing angle of 20.5 °. The inner α-helical segments also interact with symmetry-related copies of themselves, as do the outer segments. The double layer of tightly packed, intricately interlocked α-helices forms a stable, 20 Å thick protein coat around the viral DNA.     

O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1

Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C(1)XSX(A/P)C(2). We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.     

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D)¹H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs ɛ-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His³, Trp⁷², Tyr⁴¹, Tyr⁵⁰ and Tyr⁷⁴) appear to be exposed and accessible to 3-N-car☐ymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp²⁵ and Trp⁶² indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp⁷² ring in the presence of BASA. Moreover, a number of H^β resonances can be identified and sorted according to specific types of amino acid residues.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP 1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D) 1H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs epsilon-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His3, Trp72, Tyr41, Tyr50 and Tyr74) appear to be exposed and accessible to 3-N-carboxymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp25 and Trp62 indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp72 ring in the presence of BASA. Moreover, a number of H beta resonances can be identified and sorted according to specific types of amino acid residues.