Conformational studies on pertrimethylsilyl derivatives of some 6-deoxyaldohexopyranoses and of four less-common aldohexopyranoses by 220-MHz P.M.R. spectroscopy. Substituent and configurational effects on the chemical shifts of the ring protons

The complete interpretation of 220-MHz p.m.r. spectra and the accurate chemical shifts and coupling constants, obtained after computer simulation of the spectra, of the per-O-trimethylsilyl (Me₃Si) derivatives of a number of 6-deoxy-aldohexopyranoses and of β-D-altro-, β-D-allo-, and α- and β-D-talo-pyranose are given. By means of an adapted Karplus equation, the structure of the derivatives has been studied in detail. All of the pyranoid rings occur in the ⁴C₁(D) or ¹C₄(L) chair conformation. The preferred conformation of the C-5—CH₂OSiMe₃ group in the four aldohexopyranoses was found to be dependent on the configuration at C-4. By comparison of Me₃Si-aldohexopyranoses with the corresponding 6-deoxy analogues, it was found that the 6-OSiMe₃ group has no marked effect on the conformation of thering. The influence of this group on the chemical shifts of the ring protons is discussed in terms of electric field and inductive effects. Rules are presented for the estimation of the chemical shifts of the ring protons of Me₃Si-aldohexopyranoses and Me₃Si-6-deoxyaldohexopyranoses.     

A Proton Magnetic Resonance and a Circular Dichroism Study of the Solvent Dependent Conformation of the Synthetic Tubulin Fragment Ac Tubulin,Alpha (430–441) Amide and its Interaction with Substance-P

Abstract

Proton magnetic resonance techniques were used to study the conformation of the synthetic tubulin fragment Ac-tubulin (430–441) amide in H₂0 and 80% CD₃OH/20% D₂0 solutions, using water suppression techniques. Proton assignments are based on two-dimensional COSY experiments combined with one-dimensional spin decoupling.

A comparison of the NH proton shifts between the two solvents, namely ?(CD₃OH/H₂0-H₂O) shows a small solvent effect for the Lys¹ to Val⁶ region of the molecule, whereas for Gly⁷ to Glu¹² the solvent effect is much larger. The smaller effects in the region of Lys¹ to Val⁶ may be due to some hydrogen bonding as these protons are shielded from the solvent These conclusions are in agreement with the circular dichroism results in 80% methano1/20% water where the a helix is present to the extent of 30%, whereas the peptide is completely unstructured in water with some aggregation.

The temperature dependence of the NH proton shifts was also carried out. In water these shifts are of the order of7-9 × 10^?3 ppm/K indicating that most of the protons are not involved in hydrogen bonding. In CD₃0H/H₂0, these values range from about 4–6 × 10^?3 ppm/K, which are compatible with the presence of hydrogen bonds.

Finally, binding studies were carried out between the tubulin peptide and the undecapeptide neurotransmitter substance P. The largest shifts are for the Tyr³ NH proton of the tubulin fragment, whereas for substance P it is for the Lys³, Gin⁵ and Leu¹⁰ NH protons, indicating a change in conformation of both peptides on interaction.     

Approaches for the measurement of solvent exposure in proteins by <Superscript>19</Superscript>F NMR

Fluorine NMR is a useful tool to probe protein folding, conformation and local topology owing to the sensitivity of the chemical shift to the local electrostatic environment. As an example we make use of ¹⁹F NMR and 3-fluorotyrosine to evaluate the conformation and topology of the tyrosine residues (Tyr-99 and Tyr-138) within the EF-hand motif of the C-terminal domain of calmodulin (CaM) in both the calcium-loaded and calcium-free states. We critically compare approaches to assess topology and solvent exposure via solvent isotope shifts, ¹⁹F spin–lattice relaxation rates, ¹H–¹⁹F nuclear Overhauser effects, and paramagnetic shifts and relaxation rates from dissolved oxygen. Both the solvent isotope shifts and paramagnetic shifts from dissolved oxygen sensitively reflect solvent exposed surface areas.     

Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D₂O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N$\text{H}$ protons which are shifted by the metal ion are observed in the ¹H spectrum of the complex in H₂O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N^π, the carbamoyl oxygen, the pyrimidine N₁ and/or the secondary amino group may be coordinated to the iron(II).     

Trans-bilayer pseudocontact shifts produced by lanthanide ions in the 1H and 31P-nmr spectra of phospholipid vesicular membranes and their temperature variation

1. Shifts in the ¹H and ³¹P-nmr signals originating from the outer and inner phosphorylcholine head-groups and from the lipid acyl chains are observed when phosphatidylcholine vesicles are treated with increasing extravesicular concentrations of the lanthanides Eu³⁺, Pr³⁺, Yb³⁺, and Dy³⁺. 2. The addition of KNCS to increase the binding of the lanthanide ions to the outer head-groups is used to demonstrate that the intravesicular group shifts are not caused by bulk susceptibility effects. 3. The magnitude and direction of the observed shifts in the ¹H-nmr spectrum are shown to be consistent with (a) pseudocontact interaction of the paramagnetic lanthanide ions with the outer phospholipid head-groups, (b) current views of the conformation of the phosphatidylcholine head-group in the presence of lanthanides, and (c) a conservation of magnetic field within the vesicles due to their spherical nature. 4. Variation of the shifts with temperature are compared for egg phosphatidylcholine and dipalmitoyl phosphatidylcholine. The temperature variation in shifts is also used to study phase transitions in each monolayer and phase separations in mixed lipid systems.     

Conformation of adenosine 3′,5′-monophosphate in solution as studied by the NMR-desert method II. Self-association and temperature-dependent glycosidic isomerization at pH 7

A study has been made of the association and the temperature-dependent conformation of adenosine 3′,5′-monophosphate (cyclic AMP) in a neutral aqueous (²H₂O) solution by means of proton magnetic resonance chemical shift and relaxation. The concentration and temperature-dependent chemical shifts of H(1′), H(2), and H(8), have enabled us to estimate the self-association constant, K_a = 1.1 ± 0.3 M^?1 at 25°C and thermodynamic parameters ΔH = ?5.8 ± 1.5 kcal/mol and ΔS (25°C) = ?19.0 ± 3 cal/mol per degree.The NMR-DESERT (Deuterium Substitution Effect on Relaxation Times) method has been utilized for the determination of the syn-anti conformational equilibrium in the monomeric state and for the determination of the mutual orientation of the two adenine rings in the dimeric state of cyclic AMP. The molecules were found to coexist with nearly equimolarity of syn-anti conformers and thermal activation of the molecules perturbs the syn-anti conformational equilibrium to comprise the syn form in preference at higher temperature. The glycosidic isomerization (from anti to syn) was found to be characterized both by a positive enthalpy change and by a positive entropy change. The cyclic AMP molecules prefer to take a ‘trans-stacking’ conformation in the dimeric state where the two molecules are arranged in such a way that the H(2) of one molecule is close to the H(8) of the other.     

Flexibility of DNA and RNA upon Binding to Different Metal Cations. An Investigation of the B to A to Z Conformational Transition by Fourier Transform Infrared Spectroscopy

Abstract

The interaction of DNA and RNA with Cu(II), Mg(II), [Co(NH₃)₆]³⁺ [Co(NH₃)₅Cl]²⁺ chlorides and, cis- and trans-Pt(NH₃)₂Cl₂ (CIS-DDP, trans-DDP) has been studied by Fourier Transform Infrared (FT-IR) spectroscopy and a correlation between metal-base binding and conformational transitions in the sugar pucker has been established. It has been found that RNA did not change from A-form on complexation with metals, whereas DNA exhibited a B to Z transition. The marker bands for the A-form (C′₃-endo-anti conformation) were found to be near 810–816 cm^?1, while the bands at 825 and 690 cm^?1 are marker bands for the B- conformation (C′₂-endo, anti), The B to Z (C₃′-endo, syn conformation) transition is characterized by the shift of the band at 825 cm^?1 to 810–816 cm^?1 and the shift of the guanine band at 690 cm^?1 to about 600–624 cm^?1.     

Backbone Structure Confirmation and Side Chain Conformation Refinement of a Bradykinin Mimic BKM-824 by Comparing Calculated 1H, 13C and 19F Chemical Shifts with Experiment

Abstract

Calculated and experimental ¹H, ¹³C and ¹⁹F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava¹-Igl²-Ser³-DF5F⁴-Oic⁵-Arg⁶] (Ava=5-amino- valeric acid, Igl=α-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)- octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947–955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6–31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6–311G** basis set. There is an intramolecular hydrogen bond at Ser³ in the optimized structures.

The experimental ¹³C chemical shifts at five C^α positions as well as at the C^β, C^γ and Cδ position of Ava¹, which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental H^β chemical shifts in Igl² shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the χ1 angle in DF5F⁴ suggest that χ1 ≈180°. The calculated ¹H and ¹³C chemical shifts are in good agreement with experiment at the rigid residue Oic⁵. None of the models gives accurate results for Arg⁶, presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.     

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm^–1) to the lower wavelength (1457 cm^–1) CH₂ bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.Abbreviations PS phosphatidylserine natural - DMPS dimyristoyl phosphatidylserine - DPPC dipalmitoyl phosphatidylcholine - FTIR Fourier transform infrared spectroscopy - DSC differential scanning calorimetry - PE phosphatidylethanolamine Offprint requests to: D. Bach     

Spectroscopic studies on the structure of poly(8-bromoadenylic acid): Effect of glycosidic torsion angle on the conformation and flexibility in polyribonucleotides

The helix–coil transition and conformational structure of poly(8-bromoadenylic acid) [poly(8BrA)] have been investigated using ¹H- and ¹³C-nmr, CD, and ir spectroscopy. The results have been compared with the structure of the related 5′-mono- and polynucleotides. The chemical shifts of H(2′), H(3′), C(2′), and C(3′) nmr signals show an interesting correlation with both the puckering of ribose ring and glycosidic bond torsion angle. Poly(8BrA) shows an upfield shift of the C(3′) signal and a downfield shift of the H(3′) signal compared to the chemical shifts in poly(A). These shifts are consistent with a C(3′) endo-syn conformation for poly(8BrA). A similar effect has been reported previously and is also observed here on the C(2′) and H(2′) signals when the preferred conformation is C(2′)endo-syn (e.g., in 5′-8BrAMP). The chemical-shift parameters thus act as a probe for studying syn ? anti and N ? S equilibria in solutions. The three-bond ¹H-′¹³C coupling constants between H(1′) and C(8) and C(4) have been measured in poly(8BrA) and 5′-8BrAMP and their structural implications have been discussed. The observed preference of a C(3′)endo-syn conformation for poly(8BrA), coupled with other evidence, throws doubt on the validity of a correlation previously reported whereby a syn conformation is associated with a C(2′)endo ribose pucker. The backbone conformation of randomly coiled poly(8BrA) is very similar to the structures found in polyribonucleotides: poly(A) and poly(U). All three polymers show strong preferences for the backbone angles found in RNA helices. The CD spectrum of poly(8BrA) has a striking relationship to that of poly(A). The signs of all extrema are inverted, and the magnitudes are related by a constant factor. We suggest that these differences result from a change in the angle between coupled transition moment vectors in the two polymers. Infrared spectra of poly(8BrA) in H₂O and D₂O solution are reported for the frequency range below 1400 cm^?1. The antisymmetric >PO stretching vibration is observed at an unusually low frequency in the helix (1214 cm^?1). The symmetric >PO stretch occurs at ～1095 cm^?1 but is not resolved from a ring vibration near this frequency. A conformationally sensitive band, characteristic of helical RNA structures, is observed at 817 cm^?1 and disappears when the helix is melted. This observation confirms the conclusion that ordered poly(8BrA) has a regular helical structure with an RNA backbone conformation. A stereochemical explanation is provided for the failure of poly(8BrA) (or other syn polymers) to form double helices with anti-polyribonucleotides.     

The conformation of tetrafluorinated methyl galactoside anomers: crystallographic and NMR studies

The first single-crystal X-ray diffraction study of tetrafluorinated monosaccharide derivatives is presented. Both α- and β-methyl 2,3-dideoxy-2,2,3,3-tetrafluoro-d-galactopyranoside anomers adopt the ⁴C₁ conformation. The values for the C1–O1 and C1–O5 bond lengths and the O5–C1–O1–CH₃ dihedral angles are in line with what can be expected from the anomeric and exo-anomeric effects. The chair conformations are slightly distorted, presumably due to repulsion between 1,3-diaxial C–O and C–F bonds. The asymmetric unit of both compounds contains up to three independent molecules, which differ in the conformation of the hydroxymethyl group (including in one case a ‘forbidden’ gg rotamer). The molecular packing of the β-anomer shows a clear segregation between fluorinated and hydrophilic domains, while for the α-anomer the regions of fluorine segregation are broken by interleafing of OMe groups. There is one close OH?F contact, which is likely to arise from the crystal packing. NMR studies show that the two anomers also adopt a ⁴C₁ conformation in solution (D₂O, CDCl₃).     

Pressure-dependent changes in the structure of the melittin α-helix determined by NMR

A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small -helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H—O distance of the NH—O=C hydrogen bonds decreased by 0.021 ± 0.039 Å, leading to an overall compression along the entire helix of about 0.4 Å, corresponding to a static compressibility of 6 ×10^–6 bar^–1. The backbone dihedral angles and are altered by no more than ± 3° for most residues with a negative correlation coefficient of –0.85 between _i and _i–1, indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies.     

Quantitative determination of the conformation of cyclic 3',5'-adenosine monophosphate in solution using lanthanide ions as nuclear magnetic resonance probes

The conformation of cyclic 3′,5′-adenosine monophosphate in deuterium oxide has been determined at pH 2.0 and pH 5.5, using lanthanide ions as paramagnetic nuclear magnetic resonance probes.The lanthanide ion-induced shifts in the nuclear magnetic resonance energy for a given nucleus are dependent on the geometric position of that nucleus relative to the bound lanthanide ion. As expected, these shifts are pseudocontact in origin and are consistent with axial symmetry. Analysis of the concentration dependence of the shift shows that the lanthanide ion is bound to the phosphate entity giving a 1:1 complex. Further, base stacking and other intermolecular interactions are negligible.To confirm the conformation, which is found from a computer search with the above shift data, we have measured the changes in relaxation times, T₁ and T₂, induced by binding of Gd³⁺. The geometric dependence of these relaxation effects is different from that of shifts, being dependent only on distance. The agreement of these data with the computer “shift” conformation is satisfactory.Some ³¹P nuclear magnetic resonance experiments were done to confirm the metal co-ordination position although, here, there are contact contributions to both shift and relaxation.The computer program finds the conformations that have the correct geometry to account for the shift data, by searching all possible conformations. Non-bond rotations were used as a method of changing the pucker of the phosphate and ribose rings, the position of the base being defined by a single bond rotation. The nuclear magnetic resonance data and minimum van der Waals' distances were used as “active filters” in the computer search.At both values of the pH we have found closely related families of solutions, with the pucker of the phosphate and ribose rings roughly similar to those in an approximate X-ray study of cyclic AMP. The orientation of the base varies with pH.     

Paramagnetic doping of a 7TM membrane protein in lipid bilayers by Gd3+-complexes for solid-state NMR spectroscopy

A considerable limitation of NMR spectroscopy is its inherent low sensitivity. Approximately 90 % of the measuring time is used by the spin system to return to its Boltzmann equilibrium after excitation, which is determined by ¹H-T₁ in cross-polarized solid-state NMR experiments. It has been shown that sample doping by paramagnetic relaxation agents such as Cu²⁺-EDTA accelerates this process considerably resulting in enhanced sensitivity. Here, we extend this concept to Gd³⁺-complexes. Their effect on ¹H-T₁ has been assessed on the membrane protein proteorhodopsin, a 7TM light-driven proton pump. A comparison between Gd³⁺-DOTA, Gd³⁺-TTAHA, covalently attached Cu²⁺-EDTA-tags and Cu²⁺-EDTA reveals a 3.2-, 2.6-, 2.4- and 2-fold improved signal-to-noise ratio per unit time due to longitudinal paramagnetic relaxation enhancement. Furthermore, Gd³⁺-DOTA shows a remarkably high relaxivity, which is 77-times higher than that of Cu²⁺-EDTA. Therefore, an order of magnitude lower dopant concentration can be used. In addition, no line-broadening effects or peak shifts have been observed on proteorhodopsin in the presence of Gd³⁺-DOTA. These favourable properties make it very useful for solid-state NMR experiments on membrane proteins.     

Interactions of cyclic hexapeptide cyclo(Pro-Sar-Sar)2 with small molecules

N.m.r. and c.d. spectroscopy have been used to study the interactions of cyclic hexapeptide cyclo(Pro-Sar-Sar)₂ with metal ions and ammonium ions. Cyclo(Pro-Sar-Sar)₂ was found to form complexes with Li⁺, K^?, Ba²⁺ and Cu²⁺, accompanying the conformational change into a single conformer, and the conformation of cyclo(Pro-Sar-Sar)₂ in the Li⁺-complex was different from that in the Cu²⁺-complex. These findings indicate conformational flexibility of cyclo(Pro-Sar-Sar)₂. The equilibrium constant for the complexation with Li⁺ was 2.3 × 10²l mol^?1, and cyclo(Pro-Sar-Sar)₂ adopted an asymmetric conformation in the complex. The addition of α-amino acid ester hydrochloride also caused the conformational change of cyclo(Pro-Sar-Sar)₂), but in this case it did not converge into a single conformation. This type of interaction was strengthened with aromatic α-amino acid ester hydrochloride due to the aromatic-amide interactions. Finally, the rates of exchange between unbound α-amino acid ester hydrochlorides and those complexed with cyclo(Pro-Sar-Sar)₂ were found to be different, according to the nature of α-amino acid.     

The conformation of an alamethicin in methanol by multinuclear NMR spetroscopy and distance geometry/simulated annealing

The solution conformation of the antibiotic peptide alamethicin was investigated using multi-nuclear spectroscopy and the distance geometry/simulated annealing algorithms from the program DSPACE. ¹H-, ¹³C-, and ¹⁵N-nmr chemical shifts and homonuclear ¹H coupling constants suggest that the molecule is flexible in the vicinity of Gly-11 and Leu-12. The temperature dependence of the amide proton chemical shifts indicates that there is flexibility in the middle of the 20 residue peptide and provides evidence that, at the very N-terminus, the molecule adopts a 3₁₀-helical conformation. The large differences in the ¹³C chemical shifts of the pro-R and pro-S methyls of the α-aminoisobutyric acid residues were used to constrain those residues to the right-handed helical conformation in the distance geometry/simulated annealing algorithms. A family of 24 structures was generated but did not converge to a common conformation when superimposed over the entire polypeptide sequence. The molecules did converge to a helical conformation over residues 1–10 and residues 13–18. The lack of convergence when the entire lengths of the molecules are superimposed is explained by the flexibility of the peptide near Gly-11/Leu-12. The results suggest that the protein consists of two helices connected by a flexible “hinge.” The flexibility of the molecule is discussed with respect to the macrodipole model of voltage gating. © 1995 John Wiley & Sons, Inc.     

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.     

Pressure-dependent <Superscript>13</Superscript>C chemical shifts in proteins: origins and applications

Pressure-dependent ¹³C chemical shifts have been measured for aliphatic carbons in barnase and Protein G. Up to 200 MPa (2 kbar), most shift changes are linear, demonstrating pressure-independent compressibilities. CH₃, CH₂ and CH carbon shifts change on average by +0.23, −0.09 and −0.18 ppm, respectively, due to a combination of bond shortening and changes in bond angles, the latter matching one explanation for the γ-gauche effect. In addition, there is a residue-specific component, arising from both local compression and conformational change. To assess the relative magnitudes of these effects, residue-specific shift changes for protein G were converted into structural restraints and used to calculate the change in structure with pressure, using a genetic algorithm to convert shift changes into dihedral angle restraints. The results demonstrate that residual ¹³Cα shifts are dominated by dihedral angle changes and can be used to calculate structural change, whereas ¹³Cβ shifts retain significant dependence on local compression, making them less useful as structural restraints.     

Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

Summary Nearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural ¹³C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH₂ side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, ¹³C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.