Biosynthetic site-specific <Superscript>13</Superscript> C labeling of the light-harvesting 2 protein complex: A model for solid state NMR structure determination of transmembrane proteins

Partly biosynthetic site-directed isotopically ¹³C enriched photosynthetic light-harvesting 2(LH2) complexes have been prepared from Rhodopseudomonas acidophila strain 10050 by using chemically labeled [1,2,3,4–¹³C], [1,4–¹³C] and [2,3–¹³C] succinic acid as a precursor in the growth medium. Two-dimensional proton driven spin diffusion (PDSD) solid state NMR correlation spectroscopy has been used to trace each individual ¹³C isotope from the labeled succinic acid precursor to its destination into the protein and into the embedded major light-absorbing bacteriochlorophyll cofactors. For both the residues of the protein and for the cofactors distinct labeling patterns have been deduced, for protein complexes prepared from [1,4–¹³C]-succinic acid or [2,3–¹³C]-succinic labeled media. All residues, except isoleucine and leucine, have been labeled almost homogeneously by the succinic acid precursor. Carbonyl carbons in the protein backbone were labeled by [1,4–¹³C]-succinic acid, while the C and C carbons of the residues were labeled by [2,3 ¹³C]-succinic acid. Leucine and isoleucine residues were labeled using a uniformly labeled amino acid mixture in the medium. The pattern labeling yields an increase of the resolution and less spectral crowding. The partial labeling technique in combination with conventional solid state NMR methods at ultra high magnetic fields provides an attractive route to resolve chemical shifts for -helical transmembrane protein structures.     

Preparation and 13C NMR study of carbonato-, bicarbonato- and oxalato-tetraaminecobalt(III) complexes

A series of diamagnetic carbonato-, bicarbonato- and oxalato-tetraaminecobalt(III) complexes have been prepared, where the amines are: ammonia, 1,2-diaminoethane (en), 1,3-diaminopropane (tn), 2,2′,2″-triaminotriethylamine (tren), and 3,3′,3″- triaminotripropylamine (trpn), and the ¹³C NMR spectra for these complexes have been examined. ¹³C NMR provides a valuable diagnostic probe for the complexes, and assignments of chemical shifts to specific carbons are in most cases readily made. Results for [Co(trpn)(H₂O)(OCO₂H)]²⁺ indicate the presence of both geometric isomers. The studies have relevance to the use of ¹³C NMR in examining metal-ligand interactions in systems of biological interest.     

N-[1-13C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

13 C NMR Chemical shifts and carbon-proton coupling constants of some furocoumarins and furochromones

The ¹³C NMR spectra of a variety of furocoumarins, dihydrofurocoumarins and furochromones are reported. The signals were assigned using carbon-proton coupling constants, ring annullation shifts, nuclear Overhauser effect considerations and shift effects caused by monothioester formation. Substituent effects on ¹³C chemical shifts and carbon-proton coupling constants are discussed. Methoxyl induced shifts of 5- and 8-substituted furocoumarins are additive, but their effects cannot be transferred to the furochromone system.     

Assignment of the 13C nuclear magnetic resonance spectrum of a short DNA-duplex with 1H-detected two-dimensional heteronuclear correlation spectroscopy.

Proton-detected 1H-13C heteronuclear correlated spectroscopy [( 1H,13C]-COSY) was used to establish relations between the carbon-13 and proton nuclear magnetic resonance chemical shifts in the hexadeoxynucleoside pentaphosphate d-(GCATGC)2. Using the previously established sequence-specific proton NMR assignments, sequence-specific assignments were thus obtained for nearly all proton-bearing carbons. This approach offers a new criterion for distinguishing between the proton NMR lines of purines and pyrimidines, based on the different proton-carbon-13 coupling constants. Furthermore, the adenine ring carbon 2 has a unique carbon-13 chemical shift, which enables a straightforward identification of the adenine C2H resonances by [1H,13C]-COSY.     

Carbon-13 as a tool for the study of carbohydrate structures,conformations and interactions

The application of ¹³C-NMR spectroscopy to problems involving the structures and interactions of carbohydrates is described. Both ¹³C-enriched and natural abundance compounds were used and some advantages of the use of the stable isotope are described. Carbon-carbon and carbon-proton coupling constants obtained from 1-¹³ C enriched carbohydrates were employed in the assignment of their chemical shifts and to establish solution conformation. In all cases studied thus far, C-3 couples to C-1 only in the β-anomers while C-5 couples to C-1 only in the α-anomers. C-6 and C-2 always couple to C-1 in both anomeric species. The alkaline degradation of glucose [1-¹³ C] to saccharinic acids was followed by ¹³C-NMR. The conversion of glucose [1-¹³ C] to fructose-1,6-bisphosphate [1,6-¹³ C] by enzymes of the glycolytic pathway was shown as an example of the use of ¹³C-enriched carbohydrates to elucidate biochemical pathways. In a large number of glycosyl phosphates the ³¹P to H-1 and ³¹P to C-2 coupling constants demonstrate that in the preferred conformation the phosphate group lies between the O-5 and the H-1 of the pyranose ring. The influence of paramagnetic Mn²⁺ ions on the proton decoupled ¹³C-NMR spectra of uridine diphosphate N-acetylglucosamine indicates that the Mn²⁺ interacts strongly with the pyrophosphate moiety and with the carbonyl groups of the uracil and N-acetyl groups.     

Determination of extracellular and intracellular enrichments of [1-C]-α-ketoisovalerate using enzymatically converted [1-C]-valine standardization curves and gas chromatography–mass spectrometry

We report a validated method for the determination of extra- and intracellular [1-¹³C]-α-ketoisovalerate ([1-¹³C]-KIV) enrichments by gas chromatography–mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-¹³C]-valine enriched standards of known composition. Slopes of [1-¹³C]-valine standardization curves (mean±SD: 0.99±0.02, n=5) and [1-¹³C]-KIV standardization curves (mean±SD: 0.98±0.01, n=7) were not significantly different. The method was applied for the determination of [1-¹³C]-KIV enrichments in plasma and tissues during [1-¹³C]-valine infusion in a piglet. [1-¹³C]-KIV enrichment could be determined±0.1 MPE (C.V. 1%), and extracellular [1-¹³C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-¹³C]-KIV enrichment.     

1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

13C-N.M.R.-spectral study of the pH behavior of reductively [13C]methylated,glycophorin a glyco-octapeptides and a related glycopentapeptide

The pH dependence of the labeled-carbon resonances of reductively [¹³C] methylated compounds tri-l-Ser, glyco-octapeptide A^M, asialoglyco-octapeptide A^M, glyco-octapeptide A^N, asialoglyco-octapeptide A^N, and a glycopentapeptide was investigated. The results are discussed relative to those previously observed for reductively [¹³C]methylated, intact glycophorins A^M and A^N, and in terms of the mode of display of the MN blood-group specificities by these related glycoproteins. The results indicated that the α-d-NeuAc groups appear to affect the pH-titration results of glyco-octapeptides A^M and A^N. Moreover, comparison of the pH-titration results for reductively [¹³C]methylated glyco-octapeptide A^M and reductively [¹³C]methylated asialoglyco-octapeptide A^M with those of a reductively [¹³C]methylated glycopentapeptide and reductively [¹³C]methylated tri-l-Ser indicated that the other carbohydrate residues present (α-d-GalNAc and β-d-Gal) may also affect the pH-titration results. The reductive-methylation modification appears to affect the chemical shifts of the carbohydrate and peptide carbon atoms of the glycopentapeptide minimally.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Dicarboxyamylose and dicarboxycellulose,stereoregular polyelectrolytes: physicochemical characterization and interaction with divalent cations

2,3-Dicarboxyamylose (DCA) and 2,3-dicarboxycellulose (DCC) have been obtained by splitting with periodate of all the C(2)C(3) bonds of amylose and cellulose, and further oxidation (with chlorite) of the corresponding polydialdehydes. Small, but reproducible, differences of ¹³C chemical shifts in dicate that DCA and DCC retained the different configuration at C-1 of the original polysaccharides, therefore being stereoisomers. The potentiometric and conductimetric titration curves of DCA and DCC and the pH-dependence of their ¹H n.m.r. spectra are those of typical polydicarboxylates. Interaction of DCA and DCC (Na salts) with divalent cations is clearly indicated by inflexions in conductimetric titration curves with Ca²⁺, Mg²⁺, Cu²⁺ and Fe²⁺, and by variation in specific optical rotation.     

First-principles electronic structure study of rhizoferrin and its Fe(III) complexes

We have determined the structure and coordination chemistry of rhizoferrin (Rf), which is a particular type of siderophore, and its Fe(III) complexes using density functional theory calculations. Our results show that the Fe(III) ion binds in an octahedral coordination, with a low-spin (S = 1/2) charge-neutral chiral complex having the largest binding energy of the investigated complexes. We have also calculated nuclear magnetic resonance parameters, such as chemical shifts for ¹H and ¹³C, and indirect nuclear spin–spin couplings for ¹H–¹H and ¹³C–¹H in free Rf and in a low-spin neutral Rf metal complex, as well as nuclear quadrupole interaction parameters, such as asymmetry parameter and nuclear quadrupole coupling constants for ¹⁴N. Our calculated values for the chemical shifts for free Rf are in excellent agreement with experimental data while the calculated NMR parameters for Fe(III) complexes are predictions for future experimental work.     

Spirostanic diosgenin precursors from Dioscorea composita tubers

The main saponin from the fresh tuber of Dioscorea composita was dioscin and from the fermented material 3-O-[α-l-rhamnopyranosyl(1→4)-β-d-glucopyranosyl]diosgenin. The ¹³C NMR chemical shifts of saponins were used in the determination of their structure. No free sapogenin was isolated from the fresh tuber.     

A conformational study of some adenosines by use of nuclear Overhauser effect

Conformations of 8-bromo-2′-[unk]-triisopropylbenzenesulfonyladenosine ([unk]) and its 3′-[unk]-isomer ([unk]) in solution have been determined by the use of intramolecular nuclear Overhauser effects in ¹H NMR spectroscopy. Compound [unk] has been proved to have a conformation in which the adenosine and benzene rings are intramolecularly stacked and compound [unk] an elongated non-stacked conformation in dimethylsulphoxide. The 5′-[unk]-acetyl derivative of [unk] has also been found to adopt the intramolecularly stacked conformation in dimethylsulphoxide, but a non-stacked one in chloroform. Coupling constants observed are discussed in connection with the conformation of the ribose moiety. The ¹³C NMR spectra have also been examined, but no effect which could be ascribed to the stacking phenomena was observed in the carbon chemical shifts.     

Structures of chlorosomes and aggregated BChlc inChlorobium tepidum from solid state high resolution CP/MAS13C NMR

Cross polarization/magic angle spinning (CP/MAS)¹³C (solid state high resolution) NMR spectra were observed for chlorosomes and BChlc aggregates. Similarity of both kinds of spectra (except for some signals assignable to proteins and lipids in chlorosomes) indicates that BChlc's in chlorosomes are present just as in synthetic BChlc aggregates. Chemical shifts for C13¹ carbonyl and C3¹ hydroxylethyl carbons indicate hydrogen bonding between them. Comparison of solution and solid state¹³C NMR chemical shifts shows the five coordinated nature of BChlc aggregates. Some chemical shift differences were attributable to ring currents shifts. Their comparisons with calculated ring current shift values predicted structures for the aggregates. Cross polarization dynamics of the CP/MAS¹³C NMR signals explored dynamic and structural nature of the BChlc aggregates.     

Isolation and derivatization of plasma taurine for stable isotope analysis by gas chromatography-mass spectrometry

A method for the isolation and derivatization of plasma taurine is described that allows stable isotope determinations of taurine to be made by gas chromatography-mass spectrometry (gc-ms). The isolation procedure can be applied to 0.1 ml of plasma: the recovery of plasma taurine was 70–80%. For gc separation, taurine was converted to its dimethylaminomethylene methyl ester derivative which could not be detected by hydrogen flame ionization, but could be monitored readily by NH₃ chemical ionization mass spectrometry. The derivatization reaction occurred partially on-column and required optimization of injection conditions. Using stable isotope ratiometry multiple ion detection, $\text{[M + 2 + H]}{}^{\mathrm{+}}\text{[M + H]}{}^{\mathrm{+}}$ ion ratio of natural abundance taurine was determined with a standard deviation of less than ±0.07% of the ratio. The [1,2-¹³C]taurine/taurine mole ratios of standard mixtures could be accurately determined to 0.001. This stable isotope gc-ms method is suitable for studying the plasma kinetics of [1,2-¹³C]taurine in infants who are at risk with respect to taurine depletion.     

13C-n.m.r. spectra of some ribitol teichoic acids

Proton-decoupled ¹³C-n.m.r. spectra were determined for D₂O solutions of several wall teichoic acids containing glycosylated ribitol 1,5-diphosphate residues and for their dephosphorylated repeating-units. Assignments were made by correlating the chemical shift values observed with those reported for isolated constituents, allowing for perturbations of the latter resonances because of the presence of O-glycosyl or phosphodiester bonds. Anomeric configurations of hexopyranosyl residues and their position of substitution on ribitol were indicated from the distinctive chemical shifts of the carbons concerned. Three-bond ¹³C³¹P couplings (6–8 Hz) were observed, and two-bond ¹³C³¹P couplings were indicated by broadened signals. The lack of resolution for the latter resonances is probably due to the heterogeneous nature of the polymers.     

Synthetic analogues of the histidine–chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex

Mg(II)–porphyrin–ligand and (bacterio)chlorophyl–ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. ¹H, ¹³C and ¹⁵N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9–5.2 ppm ¹H, 0.2–5.5 ppm ¹³C and 2.1–27.2 ppm ¹⁵N coordination shifts were measured for selectively labeled [1,3-¹⁵N]-Im, [1,3-¹⁵N,2-¹³C]-Im and [1,3-¹⁵N,1,2-¹³C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the ¹H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a–histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.Abbreviations (B)Chl a (bacterio)chlorophyll a - HMBC heteronuclear multiple bond correlation - Im imidazole - LH light-harvesting - 1-MeIm 1-methylimidazole - Mg(II)-Por Mg(II)-porphyrin macrocycle - OEP 2,3,7,8,12,13,17,18-octaethylporphyrin     

Potentiometric measurement of stability constants of complexes between fulvic acid carboxylate and Fe3+

The stability constants of complexes formed between iron (III) and fulvic acid extracted from organic manures and wastes such as urban domestic sewage sludge, farmyard manure, poultry manure and sulfitation pressmud were investigated by the potentiometric titration method in an ionic medium of 0.1 M KNO₃ at 25±1 °C. A modification of the Katchalsky's model was employed for the estimation of stability constants. The displacement of the titration curves due to presence of Fe³⁺ in FA solutions formed the basis of calculations. The weak acidic property of fulvic acids due to carboxyl groups resulted in buffering over a wide range of pH; fulvic acids were completely neutralized in the pH range of 7.00–8.85. Apparent dissociation constants (pK_APP) of weakly acidic carboxyl groups were a direct function of degree of dissociation (α_L) in the mid-range of titration curves but were non-linear at high and low α_L values. The stability constants for formation of Fe–FA complexes (log β_Fe) calculated from the titration data were in the range of 5.64–7.55, depending upon α_L and electrostatic properties of fulvic acids. The relatively high stability constants of Fe–FA complexes in comparison to those with other competing cations suggest that the Fe–FA complexes are relatively stable in a soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Size makes a difference: Chiral recognition in complexes of fenchone with cyclodextrins studied by means of NMR titration

Gibbs energies of complex formation between enantiomers of bicyclic terpenoid, fenchone, and naturally occurring cyclodextrins, βCD and γCD, were determined by means of ¹³C and ¹H nuclear magnetic resonance (NMR) titration data. These results were compared with the corresponding data obtained previously for the diastereomeric fenchone/αCD complexes. The size of the inner cavity of host molecules significantly influences stoichiometry, association constants, and enantiomeric differentiation of the studied complexes. These complementary data allow us to discuss qualitatively the influence of the host size on the guest–host interactions. A method of the simultaneous use of titration data collected for several resonances of different isotopes in the determination of association constants was worked out and thoroughly analyzed. Comparison of the results of global data analyses with weighted means of individual ones revealed that both these approaches are equally trustworthy.