Homonuclear Hartmann-Hahn Spectroscopy Research Articles

Structural determination of oligosaccharides derived from lipooligosaccharide of Neisseria gonorrhoeae F62 by chemical, enzymic, and two-dimensional NMR methods

F62 LOS of Neisseria gonorrhoeae consists of two major LOS components; the higher and smaller molecular weight (MW) components were recognized by MAbs 1-1-M and 3F11 respectively. Base-line separation of the two major oligosaccharide (OS) components from F62 LOS was achieved by Bio-Gel P-4 chromatography after dephosphorylation of the OS mixture. The structures of the two major OSs were studied by chemical, enzymatic, and 2D NMR methods [double quantum filtered COSY (DQF-COSY), delayed COSY (D-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), pure-absorption 2D NOE NMR] as well as methylation followed by GC/MS analysis. The OS component derived from the MAb 1-1-M defined LOS component was determined to have a V3-(beta-N-acetylgalactosaminyl)neolactotetraose structure (GalNAc is beta 1----3-linked to a neolactotetraose) at one of its nonreducing termini as shown below. The above pentaose is linked to a branched diheptose-KDO core in which a GlcNAc is alpha-linked. The OS component derived from the MAb 3F11 defined LOS component did not have a GalNAc residue. The rest of its structure was identical to that of the OS-1, and a neolactotetraose is exposed at its nonreducing terminus. [formula: see text]

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.

Structure of the lipophosphoglycan from Leishmania major.

The major cell surface glycoconjugate of the parasitic protozoan Leishmania major is a heterogeneous lipophosphoglycan. It has a tripartite structure, consisting of a phosphoglycan (Mr 5,000-40,000), a variably phosphorylated hexasaccharide glycan core, and a lysoalkylphosphatidylinositol (lysoalkyl-PI) lipid anchor. The structures of the phosphoglycan and the hexasaccharide core were determined by monosaccharide analysis, methylation analysis, fast atom bombardment-mass spectrometry, one- and two-dimensional 500-MHz (correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA] 1H NMR spectroscopy, and exoglycosidase digestions. The phosphoglycan consists of eight types of phosphorylated oligosaccharide repeats which have the general structure, [formula: see text] where R = H, Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3), Glcp(beta 1-3)Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3), or Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), and where all the monosaccharides, including arabinose, are in the D-configuration. The average number of repeat units/molecule (n) is 27. Data are presented which suggest that the nonreducing terminus of the phosphoglycan is capped exclusively with the neutral disaccharide Manp(alpha 1-2)Manp alpha 1-. The structure of the glycan core was determined to be, [formula: see text] where approximately 60% of the mannose residues distal to the glucosamine are phosphorylated and where the inositol is part of the lysoalkyl-PI lipid moiety containing predominantly 24:0 and 26:0 alkyl chains. The unusual galactofuranose residue is in the beta-configuration, correcting a previous report where this residue was identified as alpha Galf. Although most of the phosphorylated repeat units are attached to the terminal galactose 6-phosphate of the core to form a linear lipophosphoglycan (LPG) molecule, some of the mannose 6-phosphate residues may also be substituted to form a Y-shaped molecule. The L. major LPG is more complex than the previously characterized LPG from Leishmania donovani, although both LPGs have the same repeating backbone structure and glycolipid anchor. Finally we show that the LPG anchor is structurally related to the major glycolipid species of L. major, indicating that some of these glycolipids may have a function as precursors to LPG.

Sequential proton NMR assignments and secondary structure of the B domain of staphylococcal protein A: structural changes between the free B domain in solution and the Fc-bound B domain in crystal

The recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G (IgG), has been investigated with the use of two-dimensional proton nuclear magnetic resonance spectroscopy. All backbone and side-chain proton resonances of FB (60 amino acid residues), except the amide proton resonance of Ala2, were assigned by the sequential assignment procedures by using double-quantum-filtered correlated spectroscopy (DQF-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and nuclear Overhauser enhancement spectroscopy (NOESY). On the basis of the NOESY data, three helical regions, Glu9-His19, Glu25-Asp37, and Ser42-Ala55, were identified in the free FB in solution. Existence of two of the three helical regions, Glu9-His19 and Glu25-Asp37, in consistent with the X-ray crystallographic structure of the Fc-bound FB [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370]. By contrast, in the Fc-bound FB as revealed by the X-ray analysis, the Ser42-Glu48 segment is extended and no structural information has been available in the Ala49-Ala55 segment. We suggest that a significant conformation change is induced in the C-terminal region of FB when it is bound to the Fc portion of IgG.

Two-Dimensional NMR Spectral Study (at 500 MHz) of a Glycooctapeptide Derived from Glycophorin AM

ABSTRACT One-dimensional and two-dimensional spectral data are presented for the N -terminal glycooctapeptide derived from the transmembrane glycoprotein, glycophorin AM. Two-dimensional homonuclear Hartmann-Hahn spectroscopy (2D-HOHAHA) and two-dimensional rotating frame nuclear Overhauser enhancement spectroscopy (2D-ROESY) were used to assign many of the resonances present in the spectra, with specific emphasis placed upon the oligosaccharide units. Not only were intra residue NOE's detected, but also inter residue NOE's involving two linked carbohydrates [β-Gal(1-->3)α-GalNAc] and also between the α-GalNac-->Thr linkage pair (between GalNAc H-1 and Thr Hβ) indicating restricted motion about this glycosidic bond. Moreover, the H-1/H-2 cross-peaks in the 2D-ROESY spectra appear to be well resolved indicating some chemical shift differences for H-1 and H-2 of the three linkage GalNAc residues found in this glycooctapeptide.

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study.

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.

The complete structure of the capsular polysaccharide from Streptococcus sanguis 34

A complete structure for the capsular polysaccharide of Streptococcus sanguis 34, which is responsible for coaggregation of this bacterium with Actinomyces viscosus T14V, an important step in the formation of dental plaque, is proposed, based partly on the 1H-n.m.r. spectrum, which was assigned by 2-dimensional COSY, homonuclear Hartmann—Hahn spectroscopy, and nuclear Overhauser effects. A phosphoric diester linkage was identified from the 31P-n.m.r. spectrum, and the linkage was determined from long range 1H— 31P correlation spectroscopy. The proposed structure is supported both by methylation analysis before and after dephosphorylation and by g.l.c.—m.s. of the phosphorylated monosaccharides as their trimethylsilyl derivatives, isolated by partial hydrolysis of the polysaccharide. The structure is composed of repeating linear hexasaccharide units joined by a phosphoric diester linkage, i.e., [→PO 4 −→6)-α- d-Gal pNAc-(1→3)-β- l-Rha p- (1→4)-β- d-Glc p-(1→6)-β- d-Gal f-(1→6)-β- d-Gal pNAc-(1→3)-α- d-Gal p-(1→) n .

Conformation change of effector-region residues in antiparallel β-sheet of human c-Ha- ras protein on GDP→GTPγS exchange: A two-dimensional NMR study

The conformations of a truncated human c-Ha- ras gene product [ ras(1–171) protein] in the GDP-bound form and in the GTPγS-bound form were compared by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). As for the GDP-bound ras(1–171) protein, three NOESY cross peaks were observed in the region of 4.5–6.0 ppm, indicating a regular antiparallel β-sheet structure. On the ligand exchange from GDP to GTPγS, one of the three NOESY cross peaks disappeared and the other two cross peaks were appreciably shifted. By analysis of the effects of specific deuteration of leucine residues and the homonuclear Hartmann-Hahn spectroscopy, the antiparallel β-sheet was found to consist of residues 38–44 and residues 51–57. The conformations around Ser-39 and Leu-56 are of the regular antiparallel β-strand type in the GDP-bound state, and largely distorted in the GTPγS-bound state, which is probably related to the conformational activation of the effector region of ras proteins by ligand exchange from GDP to GTPγS.

NMR studies of the Escherichia coli trp aporepressor. Sequence-specific assignment of the aromatic proton resonances.

The resonances in the aromatic region of the 1H-NMR spectrum of the Escherichia coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear Hartmann-Hahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts, in combination with selective deuteration of the protein. Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOEs with those expected on the basis of the crystal structure of the protein [Zhang, R.-G., Joachimiak, A., Lawson, C.L., Shevitz, R.W., Otwinowski, Z. & Sigler, P.B. (1987) Nature 327, 591-597]. The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opens the way to detailed studies of the conformational effects of corepressor and operator binding.

Solution behavior and complete proton and carbon-13 NMR assignments of the coenzyme B12 derivative (5'-deoxyadenosyl)cobinamide using modern 2D NMR experiments, including 600 MHz proton NMR data

Two-dimensional (2D) NMR methods have been used to assign completely the {sup 1}H and {sup 13}C NMR spectra of the (5{prime}-deoxyadenosyl)cobinamide cation (AdoCbi{sup +}) in D{sub 2}O. Most of the {sup 1}H spectral assignments were made by using 2D homonuclear shift correlation spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), absorption-mode (phase sensitive) 2D nuclear Overhauser effect (NOE) spectroscopy, and spin-locked NOE spectroscopy (also called ROESY, for rotating-frame Overhauser enhancement spectroscopy). Most of the protonated carbon resonances were assigned by using {sup 1}H-detected heteronuclear multiple-quantum coherence (HMQC) spectroscopy. The nonprotonated carbon resonances, as well as the remaining unassigned {sup 1}H and {sup 13}C NMR signals, were assigned from long-range {sup 1}H-{sup 13}C connectivities determined from {sup 1}H-detected multiple-bond heteronuclear multiple-quantum coherence spectroscopy (HMBC). Comparison of the {sup 13}C chemical shifts and {sup 1}H NOEs of AdoCbi{sup +} with those of coenzyme B{sup 12} ((5{prime}-deoxyadenosyl)cobalamin) and its benzimidazole-protonated, base-off form indicates that the electronic properties and structure of AdoCbi{sup +} are similar to that of coenzyme B{sup 12} in the protonated, base-off form. The {sup 13}C chemical shifts of most of the carbons of AdoCbi{sup +} do not vary significantly from those of base-off, benzimidazole-protonated coenzyme B{sup 12}, indicating that the electronic environmentmore » of the corrin ring is also similar in both compounds. However, significant differences in the chemical shifts of some of the corresponding carbons of the b, d, e, and f corrin side chains in AdoCbi{sup +} and in base-off, benzimidazole-protonated coenzyme B{sub 12} indicate that the positions of these side chains may be different in AdoCbi{sup +} compared to base-off coenzyme B{sup 12}.« less

Relayed HOHAHA, a useful method for extracting subspectra of individual components of sugar chains

The ma in difficulty encountered in the analysis of NMR spectra of sugar chains is the overlap of most sugar proton resonances except for reporter groups such as anomerit proton resonances ( 1). Therefore, NMR methods for extracting the proton resonances of each sugar component from an overlapping region are very useful. In previous studies, we applied mu ltiple relayed COSY (2-5) and homonuclear Hartmann-Hahn spectroscopy (HOHAHA) (6, 7) to extract the subspectrum of each sugar component of glycolipids (8, 9)) utilizing magnetization transfer from wellresolved anomeric proton resonances. In contrast to homonuclear mu ltiple relay experiments, the HOHAHA experiment does not generate large amounts of mu ltiplequantum coherence during the m ixing period and it redistributes the entire integrated intensity of one proton among all N protons in the same spin system. Hence, besides relaxation during the m ixing time, the sensitivity of a ID HOHAHA spectrum is reduced only by a factor N relative to a conventional one-dimensional ‘H spectrum. Magnetization transfer in the HOHAHA experiment is especially efficient when all couplings are of a similar order of magn itude. For example, for sugar residues such as G lc and G lcNAc which have couplings of 6-9 Hz all a round the ring, complete subspectra can be obtained very efficiently. If one of the couplings around the sugar ring is very small, it essentially blocks the Hartmann-Hahn flow of magnetization. For example, in Ga l, Ga lNAc, and Fuc residues, magnetization is rapidly transferred among protons HI through H4, but because of the typically very small coupling constant between H4 and H5 protons ( l1.5 Hz), HOHAHA transfer to H5 is not very efficient. Moreover, any magnetization transferred to H5 is rapidly “diluted” because of further HOHAHA transfer to the usually fast relaxing H6 protons. Here we demonstrate that a combination of HOHAHA and the conventional ‘H‘H relay mechanism can be used effectively to circumvent this problem. The pulse sequence of 1 D-relayed HOHAHA is shown in F ig. 1, where magnetization is first transferred from Hl to H4 via H2 and H3 by HOHAHA using MLEV-

Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans of swarm rat chondrosarcoma. Demonstration of the structure Gal(4-O-sulfate)beta 1-3Gal beta 1-4XYL beta 1-O-Ser.

Nonsulfated, monosulfated, and disulfated glycopeptides containing the entire carbohydrate sequence of the glycosaminoglycan-specific linkage region were isolated after exhaustive enzymatic digestions of Swarm rat chondrosarcoma proteoglycans with chondroitinase ABC, papain, and Pronase. Their structures were examined by 500 MHz 1H NMR spectroscopy. The nonsulfated compound has the following structure with trace amounts of a few additional amino acids: delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser. The monosulfated compound has an ester sulfate on C-4 of the GalNAc residue and the disulfated compound has an additional hitherto unrecognized ester sulfate on C-4 of the second galactose residue which is remote from the innermost xylose. This new structure was confirmed by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The molar ratio of the isolated nonsulfated, monosulfated, and disulfated compounds was 53:37:10 based on the serine contents. Biological significance of the newly found sulfated linkage structure is discussed.

Two-dimensional NMR studies of Kazal proteinase inhibitors. 1. Sequence-specific assignments and secondary structure of turkey ovomucoid third domain.

Two-dimensional proton NMR experiments have been used to sequentially assign resonances to all of the peptide backbone protons of turkey ovomucoid third domain (OMTKY3) except those of the N-terminal alpha-amino group whose signal was not resolved owing to exchange with the solvent. Assignments also have been made for more than 80% of the side-chain protons. Two-dimensional chemical shift correlated spectroscopy (COSY), relayed coherence transfer spectroscopy (RELAY), and two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) were used to identify the spin systems of almost half of the residues prior to sequential assignment. Assignments were based on two-dimensional nuclear Overhauser enhancements observed between adjacent residues. The secondary structure of OMTKY3 in solution was determined from additional assigned NOESY cross-peaks; it closely resembles the secondary structure determined by single-crystal X-ray diffraction of OMTKY3 in complex with Streptomyces griseus proteinase B [Fujinaga, M., Read, R.J., Sielecki, A., Ardelt, W., Laskowski, M., Jr., & James, M.N.G. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4868-4872]. The NMR data provide evidence for three slowly exchanging amide protons that were not identified as hydrogen-bond donors in the crystal structure.

A method for the rapid assignment of 1H NMR spectra of oligosaccharides using homonuclear Hartmann-Hahn spectroscopy.

An NMR method is described, based on coherence transfer in the rotating frame, which allows for the rapid determination of proton resonance assignments in oligosaccharides. The composite one-dimensional spectrum of the oligosaccharide is decomposed into a subset of spectra that are derived from individual monosaccharide residues. Proton resonance assignments can be obtained from these subspectra by inspection of the multiplicities of the component resonances and by spectral simulation. The method is shown to have particular value in the assignment of proton resonances derived from primary sequence branch-point residues, which often have complex spin-coupling patterns. Other advantages of this method over conventional techniques are discussed.

Conformational changes in the oligonucleotide duples d(GCGTTGCG). d (CGCAACGC) induced by formation of a cis‐syn thymine dimer

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.

A 1H-NMR study of human interleukin-1 beta. Sequence-specific assignment of aromatic residues using site-directed mutant proteins.

Complete identification of spin systems in the aromatic region of recombinant human interleukin-1 beta has been achieved using two-dimensional homonuclear Hartmann-Hahn spectroscopy. In addition, sequence-specific assignments for the four tyrosine residues have been carried out with the help of a series of mutant proteins, obtained by site-directed mutagenesis of the cloned gene. It is shown that, for the mutant proteins investigated, either none or only local structural changes occur. The use of NMR spectroscopy to determine the structural identity of site-directed mutant proteins with respect to the wild-type protein is discussed.