Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.     

Solution conformation of the type I collagen alpha-2 chain telopeptides studied by 1H and 13C NMR spectroscopy

The high-field 1H and 13C NMR studies of the N- and C-terminal telopeptides of the alpha-2 chain of collagen were carried out in CD3OH/H2O solutions. All proton assignments are based on two-dimensional phase-sensitive COSY and ROESY experiments. The conformation of the N-telopeptide (nonamer) is predominantly extended with a small proportion of the molecules existing in a type I beta turn. The four residues involved in this turn are D3-A4-K5-G6 which is stabilized by a C = O(D3)-NH(G6) hydrogen bond. The C-terminal telopeptide is extended throughout. A model is proposed involving charge-charge and hydrophobic interactions between the extended alpha-2 chain N-telopeptide and the adjacent segments of triple-helix. A similar model is proposed for the C-telopeptide.     

Structure analysis of proximadiol (cryptomeridiol) by 13C NMR spectroscopy

The sesquiterpene diol with antispasmodic properties, earlier isolated from Cymbopogon proximus, is shown to be identical with cryptomeridiol.     

Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic alpha-helical structure from Lys2 to Leu14. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp9 are in close contact with methylene protons of SDS micelles. T1 relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp9 with Ala9, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp9 into the membrane, as well as the amphiphilic alpha-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy

We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.     

Study of hydration of cross-linked high amylose starch by solid state 13C NMR spectroscopy

Starch is subjected to chemical treatments such as cross-linking or hydroxypropylation to meet the material requirements for food uses or controlled release in the pharmaceutical industries. In this work, two types of cross-linking formulations have been employed for the preparation of high amylose starch for use as an excipient for sustained drug release. The structural differences and chain dynamics of the modified starches in the dry and hydrated states have been compared by the use of variable contact time cross polarization-magic angle spinning solid state (13)C NMR spectroscopy.     

Derivatives of C-branched monosaccharides studied by 13C NMR

13C NMR spectra of some 3-C branched D-allofuranoses and D-ribofuranoses were obtained and interpreted. The impact of attaching the alkyl substitute to the monosaccharides on chemical shifting of the adjacent carbon atoms was shown. The experimental data are useful for elucidating structures of analogous compounds by 13C NMR.     

Dynamic Structure of Proteins in Solid State. 1H and 13C NMR Relaxation Study

Abstract

Temperature dependencies of ¹H non-selective NMR T₁ and T₂ relaxation times measured at two resonance frequencies and natural abundance ^l3C NMR relaxation times T_l and T_lr measured at room temperature have been studied in a set of dry and wet solid proteins—;Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10^?11, 10^?9 and 10^?6 s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution.     

13C NMR spectroscopy of neolignans

The ¹³C NMR spectra of 15 neolignans of several structural types and two lignans were analyzed and their carbon shifts assigned. The shifts of pyrogallol ether and ethyl phenyl carbinyl ether models were used in this connection. The stereochemistry of a dimeric sideproduct in the preparation of the latter models was determined by ¹³C NMR analysis.     

Structure and dynamics of two elastin-like polypentapeptides studied by NMR spectroscopy

The structure and dynamics of two synthetic elastin-like polypentapeptides, poly(G(1)V(1)G(2)V(2)P) and poly(AV(1)GV(2)P), were studied in D(2)O and H(2)O at various temperatures by using (1)H, (2)H,(13)C, and (15)N NMR spectra, relaxations, and PGSE self-diffusivity measurement. Signal assignments were made using COSY, NOESY, HXCORR, HSQC, HMBC, and SSLR INEPT techniques. Temperature-induced conformation changes were studied using (3)J(NHCH) couplings, NOESY connectivity, chemical shifts, and signal intensities. Hydrodynamic radii were derived from self-diffusion coefficients measured by the pulsed-gradient spin-echo (PGSE) method. Selective hydration (hydrophilic or hydrophobic) was explored using NOESY and ROESY spectral methods and longitudinal and transverse (1)H relaxation of HOD and quadrupolar (2)H relaxation of D(2)O. Four different physical states were discerned in different temperature regions for both polymers: state I of a rather extended, statistically shaped and fully hydrated polymer below the critical temperature (approximately 299-300 K); state II, a relatively coiled and globular but disordered preaggregation state, developing in a rather narrow region, 300-303 K, in the case of poly(AV(1)GV(2)P) and in a broader region, overlapping with the next one, in poly(G(1)V(1)G(2)V(2)P); state III, a tightly coiled, more compact state in the region 303-313 K; and, finally, state IV, an aggregated (and eventually flocculating and sedimenting) state beyond 313 K. States II-IV coexist in varying proportions in the whole temperature range above 299 K. A structure characterized by a beta-turn stabilized by H-bonding between the Ala carbonyl and Val(2) NH groups of poly(AV(1)GV(2)P) was detected by NOESY just above the transition temperature. States II and III are progressively more stripped of their hydration sheath but retain some molecules of water confined and relatively immobilized in their coils.     

Determination of optical purity of 3,5-dimethoxybenzoyl-leucine diethylamide by chiral chromatography and 1H and 13C NMR spectroscopy

(R)-N-3,5-dinitrobenzoyl (DNB) leucine derived chiral selector was used as an HPLC chiral stationary phase for the resolution of various racemic amino acids derivatives. In this study, determination of optical purity of an amino acid derivative was performed by chiral high performance liquid chromatography and 1H and 13C NMR spectroscopy by using the DNB leucine derived chiral selector. The accuracy and precision of each optical purity value are calculated and the data are compared to each other.     

Assignments of backbone 1H, 13C, and 15N resonances and secondary structure of ribonuclease H from Escherichia coli by heteronuclear three-dimensional NMR spectroscopy.

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.     

Metabolism of [1-13C]glucose in a synaptosomally enriched fraction of rat cerebrum studied by1H/13C magnetic resonance spectroscopy

This study explored the utility of¹H and¹³C magnetic resonance spectroscopy to study a standard synaptosomally enriched fraction (P2 pellet) made from rat cerebrum. The preparations contained high concentrations of N-acetylaspartate and -aminobutyric acid and low concentrations of glutamine, indicating that they were in fact rich in neuronal cytosol. The metabolic competence of the preparation was assessed by quantitative measurements of its ability to convert [1-¹³C]glucose into lactate, glutamate, aspartate, and other metabolites under well oxygenated conditions in 30 minutes. The minimum mean glycolytic rate was 0.8 mM glucose/min and the flow through the tricarboxylic acid cycle was equivalent to 0.2 mM glucose/min.Abbreviations ppm parts per million (chemical shift scale) - NMR nuclear magnetic resonance - GABA -aminobutyric acid - PBS phosphate-buffered normal saline solution - TSP 3-trimethylsilylpropionate During the performance of these studies Dr. A.P. Burlina was on leave from Instituto di Clinica delle Malattie Nervose e Mentali, University of Padua, Padua, Italy.     

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.     

Interaction of haemoglobin and cytochrome c with aliphatic alcohols as studied by 1H NMR spectroscopy

Association of haeme proteins, haemoglobin and cytochrome c, with eight aliphatic alcohols (methanol, ethanol. two isomeric propanols and four butanols) was studied by 1H NMR spectroscopy. NMR spectra of alcohols were monitored at 60 MHz at increasing concentration of the proteins. Selective broadening of the NMR signals of individual segments of alcohols was observed only in the case of alcohol-haemoglobin systems. Its quantitative evaluation and interpretation in terms of formation of low affinity intermolecular alcohol--protein complexes led to the conclusion that haemoglobin associates with alcohol molecules in a way depending on the length and isomeric branching of the alkyl chains; in particular, the methylene and methine groups vicinal to the hydroxyl are subject to stronger immobilization than the terminal methyls or other groups. Thus, the model of hydrophobic complexes stabilized by hydrogen bonds described previously for association of bovine serum albumin with alcohols (Lubas et al., Biochemistry, 18, 4943-4951, 1979) seems to apply also to haemoglobin association. In the case of cytochrome c association, 1H NMR data alone are insufficient for structural evaluation of the mechanism of formation of the alcohol--cytochrome c complexes.     

Solid-state 13C NMR spectroscopy of a 13C carbonyl-labeled polypeptide

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For ¹³C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled ¹⁴N from the peptide bond. Here the ¹³C₁-Gly₂ site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between ¹³C and ¹⁴N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism.     

Mestranol: the assignment of its 1H and 13C NMR spectra by means of two-dimensional NMR spectroscopy, and its photochemical decomposition

The 1H- and 13C-nmr spectra of mestranol were assigned with the help of a 2 D-J-resolved, a 2D spin echo J-correlated (SECSY) and a 2D 1H-13C hetero-shift correlation experiment. The analysis of the spectra facilitated the identification of some of the photodecomposition products of mestranol. It was shown that, upon irradiation with UV-B light in water-ethanol (1:1, v/v), products are formed by oxidation of rings B and C of the steroid.     

Structure and dynamics of the potato carboxypeptidase inhibitor by 1H and 15N NMR

The solution structure and backbone dynamics of the recombinant potato carboxypeptidase inhibitor (PCI) have been characterized by NMR spectroscopy. The structure, determined on the basis of 497 NOE-derived distance constraints, is much better defined than the one reported in a previous NMR study, with an average pairwise backbone root-mean-square deviation of 0.5 A for the well-defined region of the protein, residues 7-37. Many of the side-chains show now well-defined conformations, both in the hydrophobic core and on the surface of the protein. Overall, the solution structure of free PCI is similar to the one that it shows in the crystal of the complex with carboxypeptidase A. However, some local differences are observed in regions 15-21 and 27-29. In solution, the six N-terminal and the two C-terminal residues are rather flexible, as shown by 15N backbone relaxation measurements. The flexibility of the latter segment may have implications in the binding of the inhibitor by the enzyme. All the remaining residues in the protein are essentially rigid (S2 > 0.8) with the exception of two of them at the end of a short 3/10 helix. Despite the small size of the protein, a number of amide protons are protected from exchange with solvent deuterons. The slowest exchanging protons are those in a small two-strand beta-sheet. The unfolding free energies, as calculated from the exchange rates of these protons, are around 5 kcal/mol. Other protected amide protons are located in the segment 7-12, adjacent to the beta-sheet. Although these residues are not in an extended conformation in PCI, the equivalent residues in structurally homologous proteins form a third strand of the central beta-sheet. The amide protons in the 3/10 helix are only marginally protected, indicating that they exchange by a local unfolding mechanism, which is consistent with the increase in flexibility shown by some of its residues. Backbone alignment-based programs for folding recognition, as opposite to disulfide-bond alignments, reveal new proteins of unrelated sequence and function with a similar structure.