Parameters for the Calculation of Proton NMR Chemical Shifts of Oligoribonucleotides

Abstract

A set of empirical parameters which allows the prediction of the proton NMR chemical shifts at 70 C of non-exchangeable heterobase and anomeric protons in oligoribonucleotides has been constructed. The set is based on the highly flexible nature of oligoribonucleotide single strands and the wide range of conformational states which can be populated at relatively high temperatures (70 C or greater). A pairwise subtractive procedure, using 129 ribonucleotide oligomers (all 16 dimers, all 64 trimers, 37 tetramers, and 12 pentamers), shows that significant contributions to the observed chemical shift of protons in a given nucleoside residue are made by first, second, and third neighbors on the 3′ and the 5′ sides. The majority of the neighbors cause shielding effects with the exception of some first neighbors on the 5′ side of a given residue. The magnitude of the shielding effects is greatest for the purine heterobases and follows the order A>G>C>U, with first neighbors on the 3′ side showing more pronounced effects than second neighbors and these in turn showing larger effects than third neighbors. Second neighbors on the 5′ side showed consistently greater shieldings than first neighbors, a result attributed to the deshielding effects of the first 5′ neighbor phosphate group. The parameter Tables are applied to the prediction of proton chemical shifts in one heptamer, four hexamers, and two pentamers and give average absolute differences between predicted and observed shifts less than 0.030 ppm. The parameter approach represents an excellent method of generating initial assignments of proton chemical shifts for any single strand oligoribonucleotide.     

Ab-inito quantum mechanical calculations of NMR chemical shifts in nucleic acids constituents. II. Conformational dependence of the 1H and 13C chemical shifts in the ribose

The magnetic shielding constant of the different 13C and 1H nuclei of a deoxyribose are calculated for the C2' endo and C3' endo puckerings of the furanose ring as a function of the conformation about the C4'C5' bond. For the carbons the calculated variations are of several ppm, the C3' endo puckering corresponding in most cases to a larger shielding than the C2' endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose-3' and 5' phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides. The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

The distant shielding effect in trinucleoside diphosphates

A comparative study on the proton magnetic resonance of the dinucleoside monophosphates TpT, dApT, TpdA, the trinucleoside diphosphates dApTpT, TpTpdA, and poly T has been made. The purpose of this investigation was to establish whether in a trimer the proton chemical shifts of one terminal residue are influenced by the magnetic anistropy of the other terminal residue. The conformation of the trimers was first studied and shown to the similar to that of the corresponding dimer, except that of the percentage of the lefthanded conformers in the population of the trimers is probably lower than in the population of the dimers. The methylprotons, H-6, and H-1′ of the 3′-terminal T residue in TpTpdA (or the 5′-terminal T residue in dApTpT) are found to be more upfield than the same protons on the dimer TpT up to a magnitude of about 0.1 ppm. The shielding of these protons of the terminal T(s) in these trimers is even larger than those corresponding protons in the poly(T) at the same condition. From these results, it is concluded that a distant shielding effect is exerted by the 5′-terminal. A residue (or by the 3′-terminal A residue) on the other terminal residue in the trimers studied.     

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nearest-neighbor and next-nearest-neighbor effects in the proton NMR spectra of the oligoribonucleotides ApXpG,CpXpG, CpApXpUpG,ApGpXpC, and ApGpXpCpU

The proton nmr spectra of the oligoribonucleotides in the series CpXpG, ApXpG, CpApXpUpG, and ApGpXpC (X = A, G, C, and U), together with the reference compounds CpG, ApG, CpApUpG, and ApGpC, have been measured. A complete analysis of all the nonexchangeable base protons and the ribose H-1′ protons was made. The insertion of a nucleotide X into a oligoribonucleotide led to shift changes at both nearest-neighbor and next-nearest-neighbor positions, which were rationalized in terms of the shielding abilities of the various bases. The derived shielding trends in the ApGpXpC series of compounds were successfully used to predict the chemical shifts of resonances in the related ApGpXpCpU series.     

Interaction of substrate analogs with bovine pancreatic ribonuclease A as studied by 1H nuclear magnetic resonance

The 270 MHz 1H NMR spectra of 3'-UMP and 3'-CMP were observed in the presence of a two-fold molar excess of bovine pancreatic RNase A [EC 3.1.27.5] at various pHs. For the C(5), C(6), and C(1') protons of these nucleotides, the pH profiles of chemical shifts induced by binding to RNase A were obtained by plotting the differences between chemical shifts in the presence and the absence of RNase A against pH. Such profiles were bell-shaped for the C(5) and C(6) protons of both 3'-UMP and 3'-CMP. However the profiles of C(1') protons were not bell-shaped but appeared to consist of two bell-shaped curves and reflect the dissociations of at least three ionizable groups. The observations for the C(1') protons suggest that there are at least two forms of complexes different from each other in the interaction reflecting the chemical shift of the C(1') proton. In order to clarify the interacting sites of ribonucleotides affecting the induced shift profile of the C(1') proton, the pH titration curves were observed for 3'-dCMP in the presence of RNase A. The induced shift profile was bell-shaped for the C(1') proton as well as for the C(5) proton of the base. This indicates that the interaction at the O(2')H [or O(2')] sites of ribonucleotides causes the two forms of complexes of 3'-UMP and 3'-CMP with RNase A. The interacting sites and modes were discussed with these and the pH titration curves of His-12, His-119, and Phe-120 of RNase A in the presence of a three-fold molar excess of ribonucleotides.     

NMR studies on angiotensin II: Histidine and phenylalanine ring stacking and biological activity

The conformations of angiotensin II and the antagonist [Sar¹, Ile⁸]angiotensin II in dimethylsulfoxide have been examined by high resolution proton magnetic resonance spectroscopy at 400MHz. The chemical shifts for the aromatic protons of the phenylalanine residue in angiotensin II are consistent with shielding and restricted rotation for this side-chain. The chemical shifts for the histidine C₂ and C₄ protons in angiotensin II also indicate shielding, whereas these same protons in the antagonist [Sar¹, Ile⁸]angiotensin II do not demonstrate this shielding influence. These findings suggest a stacking interaction for the histidine and phenylalanine side-chains in angiotensin II which is important for activating angiotensin receptors.     

DNA碱基质子化学位移受所在序列的五联体性质影响

根据生物大分子核磁共振数据库(BMRB)内16条单链DNA序列中的碱基特征质子的化学位移信息,分析结果表明,五联体(pentaplet)是到目前为止可以由实验数据证明的、决定中部碱基质子化学位移水平的基本单位,即DNA碱基质子的NMR化学位移受所在五联体序列的控制。以五联体中部是T碱基为例,来自化学位移的证据符合来自量子力学计算所得“5'嘧啶-嘌呤比5'嘌呤-嘧啶的顺序更稳定”的论断,表现为5'嘧啶-嘌呤侧翼顺序导致的中部T碱基质子化学位移,比5'嘌呤-嘧啶顺序δ值小0.089。对于中部碱基质子化学位移,5'侧翼二联体效应与3'侧翼二联体效应明显不同。5'侧翼序列对五联体中部碱基质子化学位移的影响从大到小,与5'序列的色散力排列顺序更相关。氢谱上A H8、A H2、G H8、T H6、C H6的位移分布顺序,与从头计算(ab initio)和δ HMON二种伴氢碳原子净电荷计算结果最为接近,相关性好。与ab initio法得到的氢原子净电荷相关性不好。二翼碱基可以对五联体中心碱基的非交换质子在8.5!的距离上产生影响,这是对NMR偶极作用距离极限的突破。DNA的质子次级化学位移不是像蛋白质那样由氢键起主导作用。以上分析为建立双链DNA碱基质子化学位移理论预测公式提供了依据。     

Predicting 15N chemical shifts in proteins using the preceding residue-specific individual shielding surfaces from φ, ψi−1, and χ1torsion angles

Empirical shielding surfaces are most commonly used to predict chemical shifts in proteins from known backbone torsion angles, phi and psi. However, the prediction of (15)N chemical shifts using this technique is significantly poorer, compared to that for the other nuclei such as (1)H(alpha), (13)C(alpha), and (13)C(beta). In this study, we investigated the effects from the preceding residue and the side-chain geometry, chi(1), on (15)N chemical shifts by statistical methods. For an amino acid sequence XY, the (15)N chemical shift of Y is expressed as a function of the amino acid types of X and Y, as well as the backbone torsion angles, phi and psi(i-1). Accordingly, 380 empirical 'Preceding Residue Specific Individual (PRSI)' (15)N chemical shift shielding surfaces, representing all the combinations of X and Y (except for Y=Pro), were built and used to predict (15)N chemical shift from phi and psi(i-1). We further investigated the chi(1) effects, which were found to account for differences in (15)N chemical shifts by approximately 5 ppm for amino acids Val, Ile, Thr, Phe, His, Tyr, and Trp. Taking the chi(1) effects into account, the chi(1)-calibrated PRSI shielding surfaces (XPRSI) were built and used to predict (15)N chemical shifts for these amino acids. We demonstrated that (15)N chemical shift predictions are significantly improved by incorporating the preceding residue and chi(1) effects. The present PRSI and XPRSI shielding surfaces were extensively compared with three recently published programs, SHIFTX (Neal et al., 2003), SHIFTS (Xu and Case, 2001 and 2002), and PROSHIFT (Meiler, 2003) on a set of ten randomly selected proteins. A set of Java programs using XPRSI shielding surfaces to predict (15)N chemical shifts in proteins were developed and are freely available for academic users at http://www.pronmr.com or by sending email to one of the authors Yunjun Wang (yunjunwang@yahoo.com).     

Nuclear magnetic spectra of self complementary decanucleotides in solution; base sequence effect on the chemical shifts of nonexchangeable protons

The aim of this study was to attempt to determine the extent to which the chemical shifts of the nonexchangeable base protons of a DNA helix depend upon the base sequence. We measured the proton NMR spectra of twelve decadeoxynucleotides in order to carry out a "statistical" treatment. In the helices, the chemical shifts were found to be determined within +/- 0.04 ppm, largely by the nearest neighbor residues on the 5'-side, and to a smaller extent by the residue on the 3'-side. The theoretical chemical shift calculations reproduced very well the polymerization shifts measured for H2 protons of adenosines if the electrostatic field effect was taken into account. A fair agreement was also obtained for H8 protons of the adenosine and guanosine residues. However, theory underestimates the polarization effects of the base protons of cytidine. This discrepancy suggests that the conformation of this residue is different in the mononucleotides relative to double helices.     

The effects of chain length on the secondary structure of oligoadenylates

The oligoadenylates (Ap)_2–4A have been studied by proton magnetic resonance (pmr) spectroscopy. All the exterior base protons and a number of the interior base proton resonance have been assigned. The results of this work showed that the adenine bases in these oligoadenylates are intramolecularly stacked at 20°C with their bases oriented preferentially in the anti conformation about their respective glycosidic bonds. The oligomers were found to associate extensively even at concentrations of 0.02 M, primarily via “end-to-end” stacking. With increasing temperature, the oligomer bases destack, but it is argued that this unfolding process cannot be described in terms of a two-state stacked versus unstacked model. Instead, the temperature dependences of the base proton chemical shifts support a base-oscillation model. The relationship between this model and the two-state model is discussed. Finally, on the basis of the chain-length dependence of the proton chemical shifts of the various adenine bases, it was concluded that subtle variations in the secondary structure of these oligomers exist with increasing chain length. Evidence is presented to show that the effects of distant base shielding are considerably smaller than what was previously estimated. The observed departures from the “extended dimer” model are attributed to differences in the relative orientations of the bases with respect to their neighbors in the oligomer.     

Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton resonances and preliminary structure analysis.

The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending.     

Ab-inito Quantum Mechanical Calculations of NMR Chemical Shifts in Nucleic Acids Constituents II Conformational Dependence of the lH and 13C Chemical Shifts in the Ribose

Abstract

The magnetic shielding constant of the different ¹³C and ¹³H nuclei of a deoxyribose are calculated for the C2′ endo and C3′ endo puckerings of the furanose ring as a function of the conformation about the C4′C5′ bond. For the carbons the calculated variations are of several ppm, the C3′ endo puckering corresponding in most cases to a larger shielding than the C2′ endo one. For the protons the calculated variations of chemical shifts are all smaller than 1.3 ppm, that is of the order of magnitude of the variation of the geometrical shielding produced on these protons by the other units of a DNA double helix, with a change of the overall structure of the helix. The computations carried out on the deoxyribose ?3′ and 5′ phosphates for several conformations of the phosphate group tend to show that the changes of conformation of the charged group of atoms produce chemical shift variations smaller than the two conformational parameters of the deoxyribose itself. The calculations carried out for a ribose do give the general features of the differences between the carbon and proton spectra of deoxynucleosides and nucleosides.

The comparison of the measured and calculated phosphorylation shifts tend to show that the counterion contributes significantly, for some nuclei of the deoxyribose, to the shifts measured. The calculated magnitude of this polarization effect on carbon shifts suggests a tentative qualitative interpretation of carbon spectra of the ribose part of DNA double helices.     

Assignment of phosphorus-31 and nonexchangeable proton resonances in a symmetrical 14 base pair lac pseudooperator DNA fragment

The 31P chemical shifts of all 13 phosphates and the chemical shifts of nearly all of the non-exchangeable protons of a symmetrical 14 base pair lac pseudooperator DNA fragment have been assigned by regiospecific labeling with oxygen-17 and two-dimensional NMR techniques. At 22 degrees C, 8 of the 13 phosphorus resonances can distinctly be resolved while the remaining 5 resonances occur in two separate overlapping regions. The 31P chemical shifts of this particular 14 base pair oligonucleotide do not follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence the more upfield the 31P resonance occurs, as shown from other 31P assignment studies. Failure of this general rule is believed to be a result of helical distortions that occur along the oligonucleotide double helix, on the basis of the analysis of Callidine [Callidine, C.R. (1982) J. Mol. Biol. 161, 343-352]. Notable exceptions to the phosphate position relationship are 5'-Py-Pu-3' dinucleotide sequences, which resonate at a lower field strength than expected in agreement with similar results as reported by Ott and Eckstein [Ott, J., & Eckstein, F. (1985) Biochemistry 24, 253]. A reasonable correlation exists between 31P chemical shifts values of the 14-mer and the helical twist sum function of Calladine. The most unusual 31P resonance occurs most upfield in the 31P spectrum, which has been assigned to the second phosphate position (5'-GpT-3') from the 5' end. This unusual chemical shift may be the result of the predicted large helical twist angle that occurs at this position in the 14-mer sequence. Further, it is believed that the large helical twist represents a unique structural feature responsible for optimum binding contact between lac repressor protein and this 14-mer lac pseudooperator segment. Assignments of proton resonances were made from two-dimensional 1H-1H nuclear Overhauser effect (NOESY) connectivities in a sequential manner applicable to right-handed B-DNA, in conjunction with two-dimensional homonuclear and heteronuclear J-correlated spectroscopies (1H-1H COSY and 31P-1H HETCOR). Most nonexchangeable base proton and deoxyribose proton (except for some unresolved H4', H5', and H5" protons) resonances were assigned.     

1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.     

Proton nuclear magnetic resonance spectroscopy of human transferrin N-terminal half-molecule: titration and hydrogen-deuterium exchange

The binding of Ga(III) to the proteolytically derived N-terminal half-molecule of human transferrin (HTF/2N) was studied by proton nuclear magnetic resonance spectroscopy. The pH-dependent titration curves of the histidinyl C(2) proton chemical shifts were altered upon formation of the GaIIIHTF/2N(C2O4) ternary complex. Two high-pK'a histidines failed to titrate when the metal and synergistic anion formed a complex with the protein. These results implicated two histidinyl residues as direct ligands to the metal. The rates of hydrogen-deuterium exchange for the C(2) protons of certain histidinyl residues were substantially decreased by metal ion binding. The two ligand histidines were protected from exchange, and a third, low-pK'a, histidinyl residue was protected. We propose that this third histidinyl residue is involved in anion binding and may serve as the base in the putative proton-relay scheme proposed for complex formation.     

Aromatic interactions in model peptide β-hairpins: ring current effects on proton chemical shifts

Crystal structures of eight peptide β-hairpins in the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spacial proximity, with the centroid-centroid distance (R(cen)) of 4.4-5.4 ? between the two phenyl rings. Proton NMR spectra in chloroform and methanol solution reveal a significant upfield shift of the Phe(7) C(δ,δ') H(2) protons (6.65-7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometrics observed in crystals for all eight peptides. Only one of the C(δ,δ') H proton lies in the shielding zone with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations, which resemble crystal state orientation, may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe side chains provide support for close similarity between the solid state and solution conformation. Temperature dependence of aromatic ring proton chemical shift and line widths for peptide 6 (Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-(D)Pro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustment about C(α)-C(β) (χ(1) ) and C(β)-C(γ) (χ(2) ) bonds of both the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of the specific aromatic ring orientation in organic solvents provides evidence for significant stabilizing interaction.     

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.     

Investigation of the relationship between tyrosyl residues and the adenosine 5'-monophosphate binding site of rabbit liver fructose-1,6-biphosphatase as studied by chemical modification and nuclear magnetic resonance spectroscopy

The effects of AMP, fructose 6-phosphate (Fru-6-P), fructose 2,6-bisphosphate (Fru-2,6-P2), and paramagnetic ions on the aromatic region of the proton nuclear magnetic resonance (NMR) spectrum of rabbit liver fructose-1,6-bisphosphatase have been investigated at 300 MHz. Two well resolved peaks in this region of the NMR spectrum are assigned to the protons from the aromatic ring of a tyrosyl residue of the enzyme by chemical modification with tetranitromethane and by nuclear Overhauser effects. Nitration of the tyrosyl residue causes desensitization of the enzyme to AMP inhibition as well as the loss of activity. In the presence of AMP during the modifications, 1 tyrosyl residue could be protected, presumably the one observed by NMR. Binding of AMP, an allosteric inhibitor of the enzyme, to rabbit liver fructose-1,6-bisphosphatase leads to an upfield shift of the tyrosyl proton signals in the NMR spectrum. No chemical shift or line broadening could be detected in the presence of the paramagnetic manganous ion, Fru-2,6-P2, or Fru-6-P. The negative intramolecular nuclear Overhauser effect from the ribose H2' proton to the adenine H8 proton of AMP suggested that AMP binds to the enzyme with an anti conformation about the glycosidic bond. The failure to observe intermolecular nuclear Overhauser effects between the tyrosyl residue and the protons of AMP indicates that the distances between them are greater than 4 A. On the basis of these observations, it is suggested that the AMP-related tyrosyl residue may be close to the AMP binding site, but it is not directly involved in ligand binding. Rather, the protection of this tyrosyl residue by AMP as observed by chemical modification experiments may well be due to a conformational change that results from covalent modification of the enzyme.     

Synthèse et étude R.M.N. de disaccharides et trisaccharides dans la série du l-rhamnose

Various di- and tri-saccharides containing l-rhamnose were synthesized by condensation of 2,3,4-tri-O-acetyl- or 2,3,4-tri-O-benzoyl-α-l-rhamnopyranosyl bromide with an unblocked glycopyranoside. The determination of the anomeric configuration of l-rhamnose saccharides by n.m.r. is difficult because structure has a greater effect on the spectra than does configuration. The α and β configurations and the position of the substitution may be assigned from the chemical shifts of H-5 and CH₃. In all the compounds having a β configuration, a shielding of the methyl group and a deshielding of the H-5 proton have been observed as compared to the compounds having an α configuration. The H-5 proton and the methyl group of peracetylated, (1→3)-linked α-l derivatives always resonate at higher fields than the corresponding protons of (1→6)-linked α-l derivatives.