Qualitative aspects of hydrogen-deuterium exchange in the 1H, 13C, and 15N nuclear magnetic resonance spectra of viomycin in aqueous solution.

The 1H, 13C, and 15N high field nuclear magnetic resonance spectra of the cyclic peptide viomycin have been fully assigned using homo- and heteronuclear double resonance experiments and pH effects. In addition it is shown how the two- and three-bond H-D isotope effects upon carbonyl resonances may assist in their assignment. The resistance to exchange with solvent water of the amide proton involved in the transannular hydrogen bond is observed directly in the 1H spectra, via the isotope effect on a carbonyl resonance in the 13C spectra, and via the one-bond 1H couppling in the 15N spectra.     

Complete assignment of carbon signals in a stereospecific peptide via selective and single off-resonance proton decoupling experiments. Analysis of the carbon-13 nuclear magnetic resonance spectrum of alumichrome at 67.88 MHz.

Polypeptides and proteins in native conformation exhibit 13C NMR spectra which are highly nondegenerate. Assignment of resonances to carbons in particular residues is hence a prerequisite for a structural analysis of the spectroscopic data. For nonprotonated carbonyl carbons, the assignment can be achieved by selective (1H alpha)13C' 2J decoupling. Using this method, we have assigned the Orn1 and Gly2 carbonyl resonances in alumichrome at 67.9 MHz. We show that a single off-resonance experiment with the decoupling frequency centered in the aliphatic proton spectrum is sufficient to assign unequivocally all the protonated carbon resonances via analysis of the reduced 1J heteronuclear splittings. Alumichrome thus becomes the first complex polypeptide spin system whose 1H, 15N, and now 13C nuclear resonances have been fully identified to date. 13C chemical shifts and 1H--13C spin--spin couplings are discussed in terms of structural strain leading to specific orbital hybridizations and on the basis of polarization effects due to electron density shifts toward hydrogen-bonding and metal-binding sites. A number of 3J(13C--C--C--1H) coupling constants measured on selected multiplets after resolution enhancement were used to derive the x-related Karplus relationship 3J(theta) = (10.2 cos2 theta -- 1.3 cos theta + 0.2) Hz.     

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].     

Direct amide <Superscript>15</Superscript>N to <Superscript>13</Superscript>C transfers for solid-state assignment experiments in deuterated proteins

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only ¹H–¹⁵N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than ¹H–¹³C CP transfers, and employs a dipolar version of the INEPT experiment for N–C transfer. By avoiding H^N–C (H^N stands for amide protons) and C–C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired ¹³C spins.     

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

The assignment of carbonyl resonances in 13C-n.m.r. spectra of peracetylated mono- and oligo-saccharides containing D-glucose and D-mannose: an alternative method for structural determination of complex carbohydrates

In the present study, proton homonuclear (COSY) and 13C-1H heteronuclear shift-correlation, n.m.r. spectroscopies have been used to assign the carbonyl carbon resonances of peracetylated D-gluco- and D-mannopyranose monosaccharides and oligosaccharides containing residues of parent D-glucopyranose monomers. Chemical shifts of these assigned resonances, particularly those arising from acetyl groups near to aglycon substitution sites, were found to be sensitive to the position and configuration of glycosidic linkages present. In addition, evidence is presented that indicates that the shifts of these carbonyl carbon resonances depend on long-range interactions with other peracetylated pyranose monomers resulting from folding of the oligosaccharide chain. These results suggest that carbonyl carbon resonances of peracetylated carbohydrates may be useful probes of oligosaccharide structure.     

1H and 13c nmr analysis of lycorine and α-dihydrolycorine

-The ¹H and ¹³C NMR spectra of lycorine and its α-dihydro derivative have been studied. The employment of nuclear magnetic double resonance, nuclear Overhauser effect and acetylated derivatives, allows the assignment of all proton resonances. The assignments of the carbon shifts have been obtained by means of proton noise decoupled, single frequency off-resonance decoupled, single frequency selective decoupling, time dependence nuclear Overhauser effect and by comparison with reference compounds.     

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution range of both NH and C alpha H protons in this largely alpha-helical protein. We demonstrate that the combined use of four new types of heteronuclear 3D NMR spectra together with the previously described HOHAHA-HMQC 3D experiment [Marion, D., et al. (1989) Biochemistry 28, 6150-6156] can provide unambiguous sequential assignment of protein backbone resonances. Sequential connectivity is derived from one-bond J couplings and the procedure is therefore independent of the backbone conformation. All the new 3D NMR experiments use 1H detection and rely on multiple-step magnetization transfers via well-resolved one-bond J couplings, offering high sensitivity and requiring a total of only 9 days for the recording of all five 3D spectra. Because the combination of 3D spectra offers at least two and often three independent pathways for determining sequential connectivity, the new assignment procedure is easily automated. Complete assignments are reported for the proton, carbon, and nitrogen backbone resonances of calmodulin, complexed with calcium.     

Assignment of the natural abundance 13C spectrum of proteins using 13C 1H-detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon-hydrogen coupling constants.

Proton-detected heteronuclear multiple-bond 1H-13C correlations (HMBC) previously have been used for assignment purposes in a variety of isotopically enriched proteins. In the present study it is demonstrated that the technique yields an almost complete assignment of the natural abundance 13C spectrum of the protein basic pancreatic trypsin inhibitor (BPTI). In addition, the technique permits additional 1H assignments to be made for this well-studied protein. The intensities of observed correlations permit rough estimates to be made of 2J(C,H) and 3J(C,H) coupling constants. These couplings can be used for conformational studies of both the side chains and the backbone. Intra- and interresidue coupling between C alpha H and the carbonyl carbon provides information about the backbone angles psi and phi. Side-chain conformations can be determined from both two- and three-bond carbon-hydrogen coupling constants. The present study of BPTI together with its known high-precision solution structure yields an experimental correlation between resonance intensities and secondary structure. The spectra show the potential of the method in analyzing 13C NMR spectra of nonenriched proteins. The method yields 13C NMR chemical shifts, which are versatile parameters to be used to monitor structural changes, titrations, etc.     

Natural abundance 13C-NMR study of paramagnetic horse heart ferricytochrome c cyanide complex: assignment of hyperfine shifted heme methyl carbon resonances

Hyperfine shifted heme methyl carbon resonances of paramagnetic horse heart ferricytochrome c cyanide complex (Cyt-c(CN)) have been observed for the first time in the natural abundance 13C-NMR spectrum and assigned using 1H-13C heteronuclear chemical shift correlated spectroscopy (1H-13C COSY). Individual heme methyl carbon NMR signal assignment permits a direct comparison between the hyperfine shifts of heme methyl carbon and attached methyl proton resonances which provides a useful information on the delocalization mechanism of the unpaired spin from the pi-conjugated system of porphyrin ring into the peripheral methyl side chains.     

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.     

Epidermal growth factor from the mouse. Physical evidence for a tiered beta-sheet domain: two-dimensional NMR correlated spectroscopy and nuclear Overhauser experiments on backbone amide protons

When H2O-exchanged, lyophilized mouse epidermal growth factor (mEGF) is dissolved in deuterium oxide at low pH (i.e., below approximately 6.0), 13 well-resolved, amide proton resonances are observed in the downfield region of an NMR spectrum (500 MHz). Under the conditions of these experiments, the lifetimes of these amide protons in exchange for deuterons of the deuterium oxide solvent suggest that these amide protons are hydrogen-bonded, backbone amide protons. Several of these amide proton resonances show splittings (i.e., JNH alpha-CH) of approximately 8-10 Hz, indicating that their associated amide protons are in some type of beta-structure. Selective nuclear Overhauser effect (NOE) experiments performed on all amide proton resonances strongly suggest that all 13 of these backbone amide protons are part of a single-tiered beta-sheet structural domain in mEGF. Correlation of 2D NMR correlated spectroscopy data, identifying scaler coupled protons, with NOE data, identifying protons close to the irradiated amide protons, allows tentative assignment of some resonances in the NOE difference spectra to specific amino acid residues. These data allow a partial structural model of the tiered beta-sheet domain in mEGF to be postulated.     

Solution conformation of a model hexapeptide containing RGD sequence.

The solution conformation of a model hexapeptide Asp-Arg-Gly-Asp-Ser-Gly (DRGDSG) containing the RGD sequence has been studied in DMSO-d6 as well as in aqueous solution (H2O:D2O/90:10%) by 1H NMR spectroscopy. The unambiguous identification of spin systems of various amino acid residues and sequence specific assignment of all proton resonances was achieved by a combination of two dimensional COSY and NOESY experiments. The temperature coefficient data of the amide proton chemical shifts in conjunction with the vicinal coupling constants, i.e. 3JNH-C alpha H, NOESY and ROESY results indicate that the peptide in both the solvents exists in a blend of conformers with beta-sheet like extended backbone structure and folded conformations. The folded conformers do not appear to be stabilised by intramolecular hydrogen bonding. Our results are consistent with the flexibility of RGD segment observed in the NMR studies on the protein echistatin containing the RGD motif (references 23-25).     

Two-dimensional 1H NMR studies of rat atrial natriuretic factor(1–23)

Using a variety of two-dimensional NMR methods, the ¹H NMR resonances of rat ANF(1–23) in dimethyl sulfoxide-d6 solution have been assigned. Two-dimensional phase sensitive correlated spectroscopy was used to identify protons that are scalar coupled and were also used to obtain coupling constants (³J_NH-αCH) in complicated regions of the spectra. Relayed coherence transfer experiments proved useful in identifying the connectivities between the NH and β-protons of the same amino acid residue. Finally, phase sensitive 2D NOE experiments allowed the identification of protons close in space between adjacent residues, thus providing the sequential assignments as well as conformational information. These preliminary results (chemical shifts, coupling constants, NOEs) were analyzed in terms of possible polypeptide secondary structures and were found to be consistent with a β-type structure or an averaging of solution conformations (random coil).     

Transient hydrogen bonding in uniformly ¹³C,¹⁵N-labeled carbohydrates in water

We report NMR studies of transient hydrogen bonding in a polysaccharide (PS) dissolved in water without cosolvent at ambient temperature. The PS portion of the Escherichia coli O142 lipopolysaccharide is comprised of repeating pentasaccharide units of GalNAc (N-acetyl galactosamine), GlcNAc (N-acetyl glucosamine), and rhamnose in a 3:1:1 ratio, respectively. A 105-ns molecular dynamics (MD) simulation on one pentasaccharide repeat unit predicts transient inter-residue hydrogen bonds from the GalNAc NH groups in the PS. To investigate these predictions experimentally, the PS was uniformly ¹³C,¹⁵N enriched and the NH, carbonyl, C2, C4, and methyl resonances of the GalNAc and GlcNAc residues assigned using through-bond triple-resonance NMR experiments. Temperature dependence of amide NH chemical shifts and one-bond NH J couplings support that NH groups on two of the GalNAc residues are donors in transient hydrogen bonds. The remaining GalNAc and GlcNAc NHs do not appear to be donors from either temperature-dependent chemical shifts or one-bond NH J couplings. These results substantiate the presence of weak or partial hydrogen bonds in carbohydrates, and that MD simulations of repeating units in PSs provide insight into overall PS structure and dynamics. Published 2011 Wiley Periodicals, Inc. Biopolymers 97: 145–154, 2012.     

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.     

Resolution enhanced homonuclear carbon decoupled triple resonance experiments for unambiguous RNA structural characterization

Large RNAs (>30 nucleotides) suffer from extensive resonance overlap that can seriously hamper unambiguous structural characterization. Here we present a set of 3D multinuclear NMR experiments with improved and optimized resolution and sensitivity for aiding with the assignment of RNA molecules. In all these experiments strong base and ribose carbon–carbon couplings are eliminated by homonuclear band-selective decoupling, leading to improved signal to noise and resolution of the C5, C6, and C1′ carbon resonances. This decoupling scheme is applied to base-type selective ¹³C-edited NOESY, ¹³C-edited TOCSY (HCCH, CCH), HCCNH, and ribose H1C1C2 experiments. The 3D implementation of the HCCNH experiment with both carbon and nitrogen evolution enables direct correlation of ¹³C and ¹⁵N resonances at different proton resonant frequencies. The advantages of the new experiments are demonstrated on a 36 nucleotides hairpin RNA from domain 5 (D5) of the group II intron Pylaiella littoralis using an abbreviated assignment strategy. These four experiments provided additional separation for regions of the RNA that have overlapped chemical shift resonances, and enabled the assignment of critical D5 bulge nucleotides that could not be assigned using current experimental schemes.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-5093-6     

Study of the spatial structure of a cyclic analog of bradykinin in solution by two-dimensional NMR spectroscopy

H NMR resonances of [cyclo (9----18) Lys1, Gly6]bradykinin (CBK) in (CD3)2SO and H2O solution have been assigned by combined analysis of two-dimensional COSY and NOESY spectra. The presence of two slowly interchangeable conformers of CBK in (CD3)2SO is established, the minor conformer not exceeding 15% in the population. The minor conformer is absent from the aqueous solution, chemical shifts of the CBK and bradykinin NH and C alpha H protons differ insignificantly. The major CBK conformer contains at least two X-Pro trans-peptide groups and three amide protons NH Phe5, NH Arg9 and N zeta H Lys1 protected from solvent. A system of cross-peaks from the NOESY spectra of CBK in (CD3)2SO has been analysed and the maximum distance between backbone protons and neighbouring amino acid residues evaluated. The experimental data agree well with the assumed type II beta-bend in the sequence Pro2-Pro3-Gly4-Phe5. Spatial structure models for the backbone fragment 6-9 of CBK containing two intramolecular hydrogen bonds that involve the NH Arg9 and N zeta H Lys1 protons and the carbonyl groups of Phe5 and Gly4 are proposed.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.     

Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 1: NMR spectral characterization

Methyl esters of gamma-linolenic acid, alpha-linolenic acid and stearidonic acid were epoxidised using m-chloroperbenzoic acid to achieve nine cis-monoepoxy-C18 fatty acid methyl esters (FAMEs), including novel methyl cis-monoepoxy derivatives of stearidonic acid and a cis-6,7-epoxy derivative of gamma-linolenic acid. These nine monoepoxy FAMEs were purified by normal-phase HPLC, identified by LC-MS, 1H and 13C NMR, and characterized by mass spectrometry and NMR spectroscopy. This study is focused on structural characterization of these C18 monoepoxy FAMEs using techniques in NMR spectroscopy including 1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear correlation (HETCOR). The proton and carbon NMR chemical shifts of the epoxide, the double bonds, and the interrupted methylenes are assigned. Also discussed is an interpretation of the 1H and 13C NMR spectra of these monoepoxides including the changes in the 13C resonance of the olefinic carbons on the neighboring double bonds resulting from epoxide formation.