Calculation of 13C chemical shifts in rna nucleosides: structure-13C chemical shift relationships.

Isotropic 13C chemical shifts of the ribose sugar in model RNA nucleosides are calculated using SCF and DFT-GIAO ab initio methods for different combinations of ribose sugar pucker, exocyclic torsion angle, and glycosidic torsion angle. Idealized conformations were obtained using structures that were fully optimized by ab initio DFT methods starting with averaged parameters from a collection of crystallographic data. Solid-state coordinates of accurate crystal or neutron diffraction structures were also examined directly without optimization. The resulting 13C chemical shifts for the two sets of calculations are then compared. The GIAO-DFT method overestimates the shifts by an average of 5 ppm while the GIAO-SCF underestimates the shifts by the same amount. However, in the majority of cases the errors appear to be systematic, as the slope of a plot of calculated vs experimental shifts is very close to unity, with minimal scatter. The values of the 13C NMR shifts of the ribose sugar are therefore sufficiently precise to allow for statistical separation of sugar puckering modes and exocyclic torsion angle conformers, based on the canonical equation model formulated in a previous paper.

Dependence of 13C NMR chemical shifts on conformations of rna nucleosides and nucleotides.

Cross-polarization magic-angle spinning solid-state NMR spectroscopy has been used to investigate the dependence of (13)C sugar chemical shifts on specific conformational parameters of a variety of ribonucleotides and ribonucleosides. Solid-state NMR is a valuable tool for nucleoside and nucleotide structural studies since it provides the means to acquire spectra that correspond to single conformations, as opposed to (13)C solution NMR methods. The distinct effects of sugar puckering on the C1', C4', and C5' resonances of C2' endo (S type) and C3' endo (N type) furanoid conformations allow us to separate them into two groups. Further analysis of each group reveals an additional dependence of the C1' and C5' resonances on the glycosidic and C4'-C5' exocyclic torsion angles, respectively. However, it is found that the glycosidic conformation cannot independently be determined from sugar (13)C chemical shift data. The statistical methods of exploratory data analysis and discriminant analysis are used to construct two canonical coordinates-linear combinations of chemical shifts which give the statistically optimal determination of the conformation from the NMR data.

Moment analysis as a systematic tool for NMR powder pattern analysis.

The low order moments for chemical shift and second-order quadrupolar powder patterns have been calculated as functions of the anisotropy and asymmetry parameter of the governing interaction, and the expressions inverted to give these parameters as a function of the moments. Theoretical simulations and experimental experience show that moment analysis in most cases equals and in some cases exceeds the accuracy of direct inspection as a method of obtaining NMR parameters. We illustrate the efficacy of the method applied to 31P chemical shift spectra of nucleic acids, and 39K second-order patterns of series of potassium salts.

Double-resonance circuit for nuclear magnetic resonance spectroscopy.

A new double-resonance probe circuit design is described. The circuit contains no quarter-wavelength elements or equivalents, yet nonetheless achieves adequate isolation between the two input channels. It contains relatively few components, and so is both compact and efficient. It has been incorporated in two solid-state nuclear magnetic resonance (NMR) probes, with excellent results.

Intercalation complex of proflavine with DNA: structure and dynamics by solid-state NMR.

The structure of the complex formed between the intercalating agent proflavine and fibrous native DNA was studied by one- and two-dimensional high-resolution solid-state nuclear magnetic resonance (NMR). Carbon-13-labeled proflavine was used to show that the drug is stacked with the aromatic ring plane perpendicular to the fiber axis and that it is essentially immobile. Natural abundance carbon-13 NMR of the DNA itself shows that proflavine binding does not change the puckering of the deoxyribose ring. However, phosphorus-31 NMR spectra show profound changes in the orientation of the phosphodiester grouping on proflavine binding, with some of the phosphodiesters tilting almost parallel to the helix axis, and a second set almost perpendicular. The first group to the phosphodiesters probably spans the intercalation sites, whereas the tilting of the second set likely compensates for the unwinding of the DNA by the intercalator.

Solid-state 13C NMR study of tyrosine protonation in dark-adapted bacteriorhodopsin.

Solid-state 13C MAS NMR spectra were obtained for dark-adapted bacteriorhodopsin (bR) labeled with [4'-13C]Tyr. Difference spectra (labeled minus natural abundance) taken at pH values between 2 and 12, and temperatures between 20 and -90 degrees C, exhibit a single signal centered at 156 ppm, indicating that the 11 tyrosines are protonated over a wide pH range. However, at pH 13, a second line appears in the spectrum with an isotropic shift of 165 ppm. Comparisons with solution and solid-state spectra of model compounds suggest that this second line is due to the formation of tyrosinate. Integrated intensities indicate that about half of the tyrosines are deprotonated at pH 13. This result demonstrates that deprotonated tyrosines in a membrane protein are detectable with solid-state NMR and that neither the bR568 nor the bR555 form of bR present in the dark-adapted state contains a tyrosinate at pH values between 2 and 12. Deprotonation of a single tyrosine in bR568 would account for 3.6% of the total tyrosine signal, which would be detectable with the current signal-to-noise ratio. We observe a slight heterogeneity and subtle line-width changes in the tyrosine signal between pH 7 and pH 12, which we interpret to be due to protein environmental effects (such as changes in hydrogen bonding) rather than complete deprotonation of tyrosine residue(s).

Determination of the DNA sugar pucker using 13C NMR spectroscopy.

Solid-state 13C NMR spectroscopy of a series of crystalline nucleosides and nucleotides allows direct measurement of the effect of the deoxyribose ring conformation on the carbon chemical shift. It is found that 3'-endo conformers have 3' and 5' chemical shifts significantly (5-10 ppm) upfield of comparable 3'-exo and 2'-endo conformers. The latter two conformers may be distinguished by smaller but still significant differences in the carbon chemical shifts at the C-2' and C-4' positions. High-resolution solid-state NMR of three modifications of fibrous calf thymus DNA shows that these trends are maintained in high-molecular-weight DNA and confirms that the major ring pucker in A-DNA is 3'-endo, while both B-DNA and C-DNA are largely 2'-endo. The data show that 13C NMR spectroscopy is a straightforward and useful probe of DNA ring pucker in both solution and the solid state.

Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: counterion effects on the 15N shift anisotropy.

High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shift anisotropy for the Schiff base nitrogen in bR548 and bR568. Comparisons are made between all-trans-bR568 and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in 15N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR568 follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.

Solid-state 13C NMR detection of a perturbed 6-s-trans chromophore in bacteriorhodopsin.

Solid-state 13C magic angle sample spinning NMR spectroscopy has been used to study the ionone ring portion of the chromophore of bacteriorhodopsin. Spectra were obtained from fully hydrated samples regenerated with retinals 13C labeled at positions C-5, C-6, C-7, C-8, and C-18 and from lyophilized samples regenerated with retinals labeled at C-9 and C-13. C-15-labeled samples were studied in both lyophilized and hydrated forms. Three independent NMR parameters (the downfield element of the C-5 chemical shift tensor, the C-8 isotropic chemical shift, and the C-18 longitudinal relaxation time) indicate that the chromophore has a 6-s-trans conformation in the protein, in contrast to the 6-s-cis conformation that is energetically favored for retinoids in solution. We also observe an additional 27 ppm downfield shift in the middle element of the C-5 shift tensor, which provides support for the existence of a negatively charged protein residue near C-5. Evidence for a positive charge near C-7, possibly the counterion for the negative charge, is also discussed. On the basis of these results, we present a new model for the retinal binding site, which has important implications for the mechanism of the "opsin shift" observed in bacteriorhodopsin.

NMR structural analysis of a membrane protein: bacteriorhodopsin peptide backbone orientation and motion.

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved method for the synthesis of phosphatidylcholines.

An improved method for the synthesis of phosphatidylcholines from phosphatidic acid and choline is described. The technique utilizes the tetraphenylborate salt of choline together with the condensing agent 2,4,6-triisopropylbenzenesulfonyl chloride. The yields in the reaction are consistently in the range 70-75%.

Solid-state 13C NMR studies of retinal in bacteriorhodopsin.

Solid-state 13C magic-angle sample spinning (MASS) NMR has been used to study lyophilized dark-adapted purple membrane containing 13C-labeled retinals. C-10-, C-11-, and C-12-labeled derivatives each showed two lines, assigned to the coexisting 13-cis and all-trans isomers. The isotropic chemical shifts, particularly of C-11, indicate that the Schiff base is protonated. Shift anisotropies are also similar to those of model compounds, indicating that this part of the chromophore is rigid and immobile and possesses the same degree of in-plane bending as crystalline retinal derivatives. Purple membrane samples labeled on the C-19- and C-20-methyl groups both give single lines from the retinal, upfield shifted by 2.1 and 1.0 ppm, respectively, from model compounds. In all cases, high-quality spectra were obtained from approximately 50-mg samples in modest signal-averaging times. These results suggest that it is now practical to exploit the enormous potential of MASS NMR for structural studies of 13C-labeled membrane proteins.

Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

13C NMR spectra of lyophilized dark-adapted [14-13C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the 13C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called gamma effect, which results from a steric interaction between the C-14 retinal proton and the protons on the epsilon CH2 of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans, 15-anti (trans or E) and 13-cis, 15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bR568 and bR548. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with 13C shifts of the carbons adjacent to the C = N linkage in protonated ketimines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.

Solid-state nitrogen-15 nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin.

Solid-state 15N NMR has been employed to examine protonation of the Schiff base linkage in epsilon-[15N]lysylbacteriorhodopsin, the single protein in purple membrane. It is shown with spectra of model compounds that protonation of a Schiff base results in an approximate 150-ppm change in the isotropic 15N chemical shift. Concurrently, the breadth of the shift anisotropy decreases by a factor of about two from 600 to 270 ppm. The isotropic shift of the Schiff base linkage observed in dark-adapted epsilon-[15N]lysylbacteriorhodopsin closely matches those observed for the protonated model compounds, particularly the more weakly hydrogen-bonded ones. It also seems to be affected slightly by isomerization of the retinal.