H2BC: a new technique for NMR analysis of complex carbohydrates

It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem. Soc.2005, 127, 6154, Magn. Reson. Chem.2005, 43, 971-974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing (2)J(CH) coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC-TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC-TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria.     

Efficient multiple-solvent suppression for the study of the interactions of organic solvents with biomolecules

A modification of the excitation sculpting sequence [Hwang, T.-L. and Shaka, A.J. (1995) J. Magn. Reson., A112, 275–279] for achieving efficient multiple-solvents suppression in 1D and 2D ¹H NMR experiments is presented. Implementation of this scheme in the ePHOGSY experiment allows rapid and sensitive detection of weak interactions between organic solvents or small molecules and biomolecules. Application of the techniques to the peptide Sandostatin® dissolved in H₂O and DMSO is demonstrated.     

1H NMR studies of lambda cro repressor. 1. Selective optimization of two-dimensional relayed coherence transfer spectroscopy

Two-dimensional relayed coherence transfer NMR spectroscopy (RELAY) has been used to corroborate side chain spin system identities in crowded regions of the 1H NMR spectrum of the lambda cro repressor protein. The mixing time in the RELAY experiments was optimized for specific preselected spin systems by using recently developed methods [Bax, A., & Drobny, G. (1985) J. Magn. Reson, 61, 306-320], which utilize the transverse relaxation time (T2) of the molecule and relevant J couplings for the defined spin system. We demonstrate that a mixing time of 26 ms gives rise to strong C alpha H-C gamma H3 RELAY cross peaks for all valine, threonine, and isoleucine residues, while RELAY cross peaks for other spin systems are weak or are not observed. This allows for rapid and unambiguous identification of the side chain resonances for valine, isoleucine, threonine, and alanine (by elimination). The use of optimized RELAY for analyzing and identifying spin systems in complex spectra is discussed.     

Two-dimensional nMR study of bleomycin and its zinc(II) complex: reassignment of 13C resonances.

Two-dimensional NMR experiments--one bond 1H-13C correlation spectroscopy and heteronuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode--have been employed to unambiguously assign all of the 13C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous 1H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731-2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The 13C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388-396; Dabrowiak et al., (1978) Biochemistry 17, 4090-4096) but are in agreement with those reported by Akkerman et al. (1988) (Magn. Reson. Chem. 26, 793-802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous 13C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.     

Ferricytochrome c oxidation of cobaltocytochrome c. Comparison of experiments with electron-transfer theories.

Electron transfer from cobaltocytochrome c to ferricytochrome c has been studied by stopped-flow kinetics. The second-order rate constant at pH 7.0, 0.1 ionic strenght, 0.2 M phosphate, and 25 degrees C is 8.3 x 103 M-1 s-1. The activation parameters obtained from measurements made between 20 and 50 degrees C are deltaHnot equal to = 2.3 kcal mol-1 and deltaSnot equal to = -33 eu. The rate constant is not significantly dependent on ionic strength; it is also relatively independent of pH between the pK values for conformation transitions. The rate diminishes at pH greater than 12. The self-exchange reaction of cobalt cytochrome c was investigated with pulsed Fourier transform 1H NMR. The rate is too slow on the 1H NMR scale; it is estimated to be less than 133 M-1 s-1. These results together with the self-exchange rates of iron cytochrome c [Gupta, R.K., Koenig, S. H., and Redfield, A. G. (1972), J. Magn. Reson. 7, 66] were analyzed by theories of Jortner and Hopfield. The theories predict the self-exchange of Cocyt c to be too slow for 1H NMR determination. The rate constant calculated by the nonadiabatic multiphonon electron-tunneling theory for the Fecyt c-Fecyt c+ and Cocyt c-Fecyt c+ electron transfers are in good agreement with experiments.     

Sequence-specific assignment of 1H-NMR resonance and determination of the secondary structure of Jingzhaotoxin-I

Jingzhaotoxin-I （JZTX-I） purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3.The structure of this toxin in aqueous solution was investigated using 2-D ^1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the ^1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in n20 and D20. All the backbone protons except for Gin4 and more than 95% of the side-chain protons were identified by dαN,dαδ, dβN and dNN connectivities in the NOESY spectrum. These studies provide a basis for the furtherdeter mination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel β-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I （HWTX-I） and hainantoxin-IV （HNTX-IV）, whose structures in solution havepre viously been reported.     

The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.

Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F. M. (1991) J. Mol. Biol. (in press)] have demonstrated that 1H NMR chemical shifts are strongly dependent on the character and nature of protein secondary structure. In particular, it has been found that the 1H NMR chemical shift of the alpha-CH proton of all 20 naturally occurring amino acids experiences an upfield shift (with respect to the random coil value) when in a helical configuration and a comparable downfield shift when in a beta-strand extended configuration. On the basis of these observations, a technique is described for rapidly and quantitatively determining the identity, extent, and location of secondary structural elements in proteins based on the simple inspection of the alpha-CH 1H resonance assignments. A number of examples are provided to demonstrate both the simplicity and the accuracy of the technique. This new method is found to be almost as accurate as the more traditional NOE-based methods of determining secondary structure and could prove to be particularly useful in light of the recent development of sequential assignment techniques which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667]. We suggest that this new procedure should not necessarily be seen as a substitute to existing rigorous methods for secondary structure determination but, rather, should be viewed as a complement to these approaches.     

Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. 1H NMR spin systems were analyzed by conventional 2D 1H[1H] methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by 1H NMR experiments were confirmed and extended by 1H-13C and 1H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three alpha-helical domains and three antiparallel beta-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H alpha-HN connectivities revealed additional information about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3',5'-bisphosphate-Ca2+ ternary complex [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555].     

Doubly sensitivity-enhanced 3D TOCSY-HSQC

Summary Recently, strategies for double sensitivity enhancement in heteronuclear three-dimensional NMR experiments were introduced (Krishnamurthy, V.V. (1995) J. Magn. Reson., B106, 170–177; Sattler et al. (1995) J. Biomol. NMR, 6, 11–22; Sattler et al. (1995) J. Magn. Reson., B108, 235–242). Since a sensitivity enhancement of a factor 2^1/2 can be achieved for each indirect dimension, nD spectra can theoretically be enhanced up to a factor of 2^((n-1)/2). We propose and analyze a doubly enhanced three-dimensional TOCSY-HSQC sequence. The application of the doubly enhanced three-dimensional {¹⁵N, ¹H} TOCSY-HSQC sequence is shown for uniformly ¹³C-/¹⁵N- and ¹⁵N-labeled samples of the relatively large Azotobacter vinelandii flavodoxin II (179 amino acids). The main factors that contribute to the final signal-to-noise enhancement have been systematically investigated. The sensitivity enhancement obtained for the doubly enhanced TOCSY-HSQC pulse sequence as compared to the standard (unenhanced) version is close to the theoretically expected factor of two.     

Magic-angle spinning NMR studies of molecular organization in multibilayers formed by 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine.

Magic-angle spinning 1H and 13C nuclear magnetic resonance (NMR) have been employed to study 50%-by-weight aqueous dispersions of 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine (C[18]:C[10]PC) and 1-octadecanoyl-2-d19-decanoyl-PC (C[18]:C[10]PC-d19), mixed-chain phospholipids which can form interdigitated multibilayers. The 1H NMR linewidth for methyl protons of the choline headgroup has been used to monitor the liquid crystalline-to-gel (LC-to-G) phase transition and confirm variations between freezing and melting temperatures. Both 1H and 13C spin-lattice relaxation times indicate unusual restrictions on segmental reorientation at megahertz frequencies for C(18):C(10)PC as compared with symmetric-chain species in the LC state; nevertheless each chemical moiety of the mixed-chain phospholipid exhibits motional behavior that may be classified as liquidlike. Two-dimensional nuclear Overhauser spectroscopy (NOESY) on C(18):C(10)PC and C(18):C(10)PC-d19 reveals cross-peaks between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup, and several experimental and theoretical considerations argue against an interpretation based on spin diffusion. Using NMR relaxation times and NOESY connectivities along with a computational formalism for four-spin systems (Keepers, J. W., and T. L. James. 1984. J. Magn. Reson. 57:404-426), an estimate of 3.5 A is obtained for the average distance between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup. This finding is consistent with a degree of interdigitation similar to that proposed for organized assemblies of gel-state phosphatidylcholine molecules with widely disparate acyl-chain lengths (Hui, S. W., and C.-H. Huang. 1986. Biochemistry. 25:1330-1335); however, acyl-chain bendback or other intermolecular interactions may also contribute to the NOESY results. For multibilayers of C(18):C(10)PC in the gel phase, 13C chemical-shift measurements indicate that trans conformers predominate along both acyl chains. 13C Spin-lattice relaxation times confirm the unusual motional restrictions noted in the LC state; nevertheless, 13C and 1H rotating-frame relaxation times indicate that the interdigitated arrangement enhances chain or bilayer motions which occur at mid-kilohertz frequencies.     

Dynamics of β-CH and β-CH2 Groups of Amino Acid Side Chains in Proteins

The dynamics of amino acid side chains of uniformly 13C/15N-enriched ribonuclease T1 (RNase T1) have been investigated. Heteronuclear longitudinal relaxation rates, 1H/13C NOEs, and transverse cross-correlated cross-relaxation rates between the Sx and the SxIz1Iz2 operators (SIIS cross relaxation) [Ernst and Ernst (1994) J. Magn. Reson., A110, 202-213] have been determined in this study. New pulse sequences for measuring the longitudinal relaxation time and the heteronuclear NOE of aliphatic side chain carbon nuclei were developed using the CCONH type of magnetization transfer and 1HN detection. In addition, an improved pulse sequence for the determination of the SIIS cross relaxation is presented. For the analysis of the relaxation rates, the model of restricted rotational diffusion around the 1 dihedral angle has been applied [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. These techniques were used in order to describe the side chain dynamics of the small globular protein RNase T1 (104 amino acids, MW about 11 kDa). Qualitative values of microdynamical parameters were obtained for 73 out of 85 amino acid side chains (glycine and alanine residues excepted) whereas more quantitative values were derived for 67 -CH and -CH2 groups.     

Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment

Complete sequence-specific assignments of the 1H NMR spectrum of bungarotoxin were reported in the previous paper [Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., & Kuntz, I.D. (1988) Biochemistry (first paper of three in this issue)]. The assignment was significantly aided by the use of the homonuclear Hartman-Hahn relayed coherence transfer nuclear Overhauser enhancement spectroscopy experiment (HRNOESY) which we present here, as a modification of relayed coherence transfer nuclear Overhauser enhancement spectroscopy (relayed NOESY) [Wagner, G. (1984) J. Magn. Reson. 57, 497]. As shown here, HRNOESY resolves problems of proton resonance overlap especially in extended chain conformations as found in beta-sheets.     

A unified NMR strategy for high-throughput determination of backbone fold of small proteins

An efficient semi-automated strategy called PFBD (i.e. Protein Fold from Backbone Data only) has been presented for rapid backbone fold determination of small proteins. It makes use of NMR parameters involving backbone atoms only. These include chemical shifts, amide?Camide NOEs and H-bonds. The backbone chemical shifts are obtained in an automated manner from the orthogonal 2D projections of variants of HNN and HN(C)N experiments (Kumar et al., in Magn Reson Chem 50(5):357?C363, 2012) using AUTOBA (Borkar et al. in J Biomol NMR 50(3):285?C297, 2011); backbone H-bonds are manually derived from constant time long-range 2D-HnCO spectrum (Cordier and Grzesiek in J Am Chem Soc 121:1601?C1602, 1999); and amide?Camide NOEs are derived from 3D HNCO NOESY experiment which provides NOEs along the direct ¹H dimension that has maximum resolution (Lohr and Ruterjans in J Biomol NMR 9(1):371?C388, 1997). All the experiments needed for the execution of PFBD can be recorded and analyzed in about 24?C48?h depending upon the concentration of the protein and dispersion of amide cross-peaks in the ¹H?C¹⁵N correlation spectrum. Thus, we believe that the strategy, because of its speed and simplicity will be very valuable in Biomolecular NMR community for high-throughput structural proteomics of small folded proteins of MW?<?10?C12?kDa, the regime where NMR is generally preferred over X-ray crystallography. The strategy has been validated and demonstrated here on two small globular proteins: human ubiquitin (76 aa) and chicken SH3 domain (62 aa).     

A model for diffusive transport through a spherical interface probed by pulsed-field gradient NMR.

In biological systems, because of higher intracellular viscosity and/or the restriction of the diffusion space inside cells, the (apparent) diffusion coefficient of an intracellular species (e.g., water) is generally smaller than when it is in the extracellular medium. This difference affects the spin-echo signal attenuation in the pulsed field gradient NMR experiment and thus affords a means of separating the intracellular from the extracellular species, thereby providing a basis for studying transmembrane transport. Such experiments have commonly been analyzed using the macroscopic model of Kärger (see Adv. Magn. Reson. 21:1-89 (1988)). In our previous study, we considered a microscopic model of diffusive transport through a spherical interface using the short gradient pulse approximation (J. Magn. Reson. A114:39-46 (1995)). The spins in the external medium were modeled with the "partially absorbing wall" condition or as having a small but finite lifetime. In the present paper, we extend our treatment to the case in which there is no limitation upon the lifetime in either medium. We also consider a simple modification of Kärger's model that more properly accounts for the restricted intracellular diffusion. Importantly, it was found that the exact solution within the short gradient pulse approximation developed here and the modified Kärger model are in close agreement in the (experimentally relevant) long-time limit. The results of this study show that when there is no limitation upon the lifetime of the transported species in either phase, the spin-echo attenuation curve is very sensitive to transport.     

Conformational signatures of <Superscript>13</Superscript>C chemical shifts in RNA ribose

The conformational dependence of ¹³C chemical shift values of RNA riboses determined by liquid-state NMR spectroscopy was evaluated using data deposited for RNA structures in the RCSD and BMRB data bases. Results derived support the applicability of the canonical coordinates approach of Rossi and Harbison (J Magn Reson 151:1–8, 2001) in liquid-state NMR to assess the sugar pucker of ribose units in RNA. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mapping of the spectral densities of N-H bond motions in eglin c using heteronuclear relaxation experiments.

A new strategy is used for studying the internal motions of proteins based on measurements of NMR relaxation parameters. The strategy yields values of the so-called spectral density functions J(omega) for N-H bond vectors. The spectral density functions are related to the distribution of frequencies contained in the rotational (overall and internal) motions of these NH bond vectors. No a priori model assumptions about the dynamics are required in this approach. The method involves measurements of six relaxation parameters consisting of 15N longitudinal relaxation rates, transverse relaxation rates of in-phase and antiphase coherence, the relaxation rates of heteronuclear 1H-15N two-spin order, the heteronuclear 1H-15N nuclear Overhauser effects, and longitudinal relaxation rates of the amide protons. The values of the spectral density functions at the five frequencies 0, omega N, omega H + omega N, omega H, and omega H - omega N are determined from the relaxation parameters using analytical relations derived previously [Peng & Wagner (1992) J. Magn. Reson. 98, 308-332]. Here, the method is applied to characterize the backbone dynamics of the 15N-enriched proteinase inhibitor eglin c, a protein of 70 residues. The values for J(0) and J(omega N = 50 MHz) vary significantly with the amino acid sequence, whereas the spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are typically much smaller and show no significant variation with the sequence. The collective behavior of the J(omega) values indicate greater internal motion for the proteinase binding loop residues and the first eight N-terminal residues. The additional internal motion in these regions is in the rate range below 450 MHz. The values of J(omega) are also compared with root mean square deviations (rmsds) of backbone atoms as obtained in NMR structure determinations. Low values of J(0) and J(omega N) are correlated with high rmsds. Spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are small and show no correlation with rmsds. A comparison with the spectral density functions obtained by fitting the experimental data to the functional dependence of the Lipari and Szabo formalism [Lipari & Szabo (1982a) J. Am. Chem. Soc. 104, 4546-4559] is made.     

Deoxyribose ring conformation of [d(GGTATACC)]2: an analysis of vicinal proton-proton coupling constants from two-dimensional proton nuclear magnetic resonance

Exchangeable and nonexchangeable protons of [d(GGTATACC)]2 in aqueous cacodylate solution were assigned from two-dimensional nuclear Overhausser effect (2D NOE) spectra. With phase-sensitive COSY and double quantum filtered COSY (DQF-COSY) experiments, the cross-peaks resulting from deoxyribose ring conformation sensitive proton-proton vicinal couplings, i.e., all 1'-2', 1'-2", 2'-3', and 3'-4' couplings and six from 2"-3' couplings, were observed. From the cross-peak fine structure, the 2',2" proton assignments can be confirmed; coupling constants J1'2' and J1'2" and sums of coupling constants involving H2' and H2" for all residues and H3' for C8 were obtained. The DISCO procedure [Kessler, H., Muller, A., & Oschkinat, H. (1985) Magn. Reson. Chem. 23, 844-852] was used to extract individual 1'-2' and 1'-2" coupling constants. The sum of coupling constants involving H1' or H3' was measured from the one-dimensional spectrum where signal overlap is not a problem. Analysis of the resulting coupling constants and sums of coupling constants, in the manner of Rinkel and Altona [Rinkel, L. J., & Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649], led to the following conclusion: C2'-endo deoxyribose ring conformation is predominant for every residue, but a significant amount of C3'-endo conformation may exist, ranging from 14% to 30%.     

Secondary structure of human granulocyte colony-stimulating factor derived from NMR spectroscopy.

Recombinant 15N-, 13C-labeled human granulocyte colony-stimulating factor (rh-metG-CSF) has been studied by 2D and 3D NMR using uniformly labeled protein as well as residue-specific 15N-labeled samples. Assignment of the 1H, 15N backbone, and 60% 1H sidechain resonances has enabled the determination of the secondary structure of the protein. The secondary structure is dominated by alpha-helical regions with four stretches of helices between residues 11-41, 71-95, 102-124 and 144-170.     

Two-Dimensional NMR Study of Bleomycin and Its Zinc(II) Complex: Reassignment of 13C Resonances

Abstract

Two-dimensional NMR experiments-one bond ¹H- ¹³C correlation spectroscopy and hetero- nuclear multiple bond correlation spectroscopy, both performed in the reverse detection mode-have been employed to unambiguously assign all of the ¹³C resonances of the antibiotic bleomycin and its zinc(II) complex. Previous ¹H resonance assignments of bleomycin (Chen et al. (1977) Biochemistry 16, 2731–2738) were confirmed on the basis of homonuclear Hartmann-Hahn and homonuclear COSY experiments. The ¹³C assignments differ substantially from those previously obtained by other investigators (Naganawa et al., (1977) J. Antibiot. 30, 388–396; Dabrowiak et al., (1978) Biochemistry 17, 4090–4096) but are in agreement with those reported by Akkerman et al.(1988) (Magn. Reson. Chem. 26, 793–802). The more recent study employed similar two-dimensional correlation experiments (performed in the direct detection mode) in conjunction with attached proton tests. Their study often required model compound data to identify carbonyls adjacent to aliphatic moieties. Previous ¹³C NMR studies of the structure, pH titration, and molecular dynamics of bleomycin and its zinc complex have been reinterpreted in terms of the revised assignments.     

Joint composite-rotation adiabatic-sweep isotope filtration

Joint composite-rotation adiabatic-sweep isotope filters are derived by combining the composite-rotation [Stuart AC et al. (1999) J Am Chem Soc 121: 5346–5347] and adiabatic-sweep [Zwahlen C et al. (1997) J Am Chem Soc 119:6711–6721; Kupče E, Freeman R (1997) J Magn Reson 127:36–48] approaches. The joint isotope filters have improved broadband filtration performance, even for extreme values of the one-bond ¹H–¹³C scalar coupling constants in proteins and RNA molecules. An average Hamiltonian analysis is used to describe evolution of the heteronuclear scalar coupling interaction during the adiabatic sweeps within the isotope filter sequences. The new isotope filter elements permit improved selective detection of NMR resonance signals originating from ¹H spins attached to an unlabeled natural abundance component of a complex in which the other components are labeled with ¹³C and ¹⁵N isotopes.