Two-dimensional Heteronuclear NMR Spectroscopy Research Articles

Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation.

The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin’s biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of β-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin’s first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin’s putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.

Detailed Chemical Composition of Condensed Tannins via Quantitative (31)P NMR and HSQC Analyses: Acacia catechu, Schinopsis balansae, and Acacia mearnsii.

The chemical composition of Acacia catechu, Schinopsis balansae, and Acacia mearnsii proanthocyanidins has been determined using a novel analytical approach that rests on the concerted use of quantitative (31)P NMR and two-dimensional heteronuclear NMR spectroscopy. This approach has offered significant detailed information regarding the structure and purity of these complex and often elusive proanthocyanidins. More specifically, rings A, B, and C of their flavan-3-ol units show well-defined and resolved absorbance regions in both the quantitative (31)P NMR and HSQC spectra. By integrating each of these regions in the (31)P NMR spectra, it is possible to identify the oxygenation patterns of the flavan-3-ol units. At the same time it is possible to acquire a fingerprint of the proanthocyanidin sample and evaluate its purity via the HSQC information. This analytical approach is suitable for both the purified natural product proanthocyanidins and their commercial analogues. Overall, this effort demonstrates the power of the concerted use of these two NMR techniques for the structural elucidation of natural products containing labile hydroxy protons and a carbon framework that can be traced out via HSQC.

Hydrothermal liquefaction oil and hydrotreated product from pine feedstock characterized by heteronuclear two-dimensional NMR spectroscopy and FT-ICR mass spectrometry

Hydrothermal liquefaction (HTL) oil and hydrotreated product from pine tree farm waste (forest product residual, FPR) have been analyzed by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) in both positive- and negative-ionization modes and high-resolution two-dimensional heteronuclear 1H–13C NMR spectroscopy. FT-ICR MS resolves thousands of compounds in complex oils and provides unparalleled compositional details for individual molecules for identification of compound class (heteroatom content), type (number of rings plus double bonds to carbon or double bond equivalents (DBE) and carbon number (degree of alkylation). Heteronuclear 1H–13C NMR spectroscopy provides one-bond and multiple-bond correlations between pairs of 1H and 13C chemical shifts that are characteristic of different organic functional groups. Taken together this information provides a picture of the chemical composition of these oils. Pyrolysis crude oil product from pine wood was characterized for comparison. Generally, pyrolysis oil is comprised of a more diverse distribution of heteroatom classes with higher oxygen number relative to HTL oil as shown by both positive- and negative-ion ESI FT-ICR MS. A total of 300 N1, 594 O1 and 267 O2 compounds were observed as products of hydrotreatment. The relative abundance of N1O1, N1O2, N1O3, N2, N2O1, N2O2 and O3 compounds are reduced to different degrees after hydrotreatment and other higher heteroatom containing species (O4–O10, N1O4, N1O5 and N2O3) are completely removed by hydrotreatment.

Measurement and Control of pH in the Aqueous Interior of Reverse Micelles

The encapsulation of proteins and nucleic acids within the nanoscale water core of reverse micelles has been used for over 3 decades as a vehicle for a wide range of investigations including enzymology, the physical chemistry of confined spaces, protein and nucleic acid structural biology, and drug development and delivery. Unfortunately, the static and dynamical aspects of the distribution of water in solutions of reverse micelles complicate the measurement and interpretation of fundamental parameters such as pH. This is a severe disadvantage in the context of (bio)chemical reactions and protein structure and function, which are generally highly sensitive to pH. There is a need to more fully characterize and control the effective pH of the reverse micelle water core. The buffering effect of titratable head groups of the reverse micelle surfactants is found to often be the dominant variable defining the pH of the water core. Methods for measuring the pH of the reverse micelle aqueous interior using one-dimensional 1H and two-dimensional heteronuclear NMR spectroscopy are described. Strategies for setting the effective pH of the reverse micelle water core are demonstrated. The exquisite sensitivity of encapsulated proteins to the surfactant, water content, and pH of the reverse micelle is also addressed. These results highlight the importance of assessing the structural fidelity of the encapsulated protein using multidimensional NMR before embarking upon a detailed structural and biophysical characterization.

Secondary organic aerosol formation from multiphase oxidation of limonene by ozone: mechanistic constraints via two-dimensional heteronuclear NMR spectroscopy

Because it is doubly unsaturated, gaseous limonene and ozone reactions exhibit considerable potential for not only a large quantity of secondary organic aerosol (SOA), but also a diverse and complicated product mixture influenced by reactant conditions. We explore the chemistry of limonene ozonolysis and provide evidence that under low-NOx conditions, the endocyclic double bond is oxidized in the gas phase, while under excess ozone conditions, the residual exocyclic double bond in condensed-phase products is heterogeneously oxidized by ozone. We use regular and multinuclear-multidimensional NMR spectroscopy, in particular 1D 1H-NMR, 1H,1H-COSY correlation spectroscopy, and 1H,13C-HSQC (heteronuclear single quantum coherence). For structural assignments we simulate 1H and 13C NMR spectra with ACDLabs online, relying representative products consistent with the postulated reaction mechanism. The 1-D 1H-NMR data allow us to quantify the extent to which the residual unsaturation is oxidized with rising ozone, confirming our hypothesis that the residual unsaturation is oxidized via heterogeneous uptake of ozone to fresh SOA particles.

Green Tea Catechins Inhibit Bacterial DNA Gyrase by Interaction with Its ATP Binding Site

Catechins are the main ingredients of green tea extracts and have been shown to possess versatile biological activities, including antimicrobial. We determined that the catechins inhibit bacterial DNA gyrase by binding to the ATP binding site of the gyrase B subunit. In the group of four tested catechins, epigallocatechin gallate (EGCG) had the highest activity, followed by epicatechin gallate (ECG) and epigallocatechin (EGC). Specific binding to the N-terminal 24 kDa fragment of gyrase B was determined by fluorescence spectroscopy and confirmed using heteronuclear two-dimensional NMR spectroscopy of the EGCG-15N-labeled gyrase B fragment complex. Protein residues affected by binding to EGCG were identified through chemical shift perturbation. Molecular docking calculations suggest that the benzopyran ring of EGCG penetrates deeply into the active site while the galloyl moiety anchors it to the cleft through interactions with its hydroxyl groups, which explains the higher activity of EGCG and ECG.

Discovery and investigation of lead compounds as binders to the Extra‐Domain B of the angiogenesis marker, fibronectin

AbstractAngiogenesis, the growth of new blood vessels from preexisting vessels, is a rare event in the adult (except in the case of the female reproductive cycle), but is a characteristic feature of diseases such as cancer, blinding ocular disorders (e.g., retinopathies), and rheumatoid arthritis. The ED‐B domain of fibronectin, a domain of 91 amino acids, inserted by a mechanism of alternative splicing of the primary transcript into the fibronectin molecule, is a high‐quality marker of angiogenesis and a target for molecular intervention, characterized by a high number of acidic residues on its surface, as well as some solvent‐exposed hydrophobic residues. A library of 113 low molecular‐weight organic compounds, containing both an aromatic moiety and at least one positive charge, was screened for binding to the ED‐B domain, using two‐dimensional heteronuclear NMR spectroscopy. One lead compound, 2,2‐diphenylethylamine, was found that binds specifically to the ED‐B domain, albeit with a dissociation constant in the millimolar range. Chemical modification of this scaffold revealed structural determinants required for binding, as well as amino acid residues in the ED‐B domain responsible for the interaction. The results presented represent the basis for the development of high‐affinity, low molecular‐weight binders by using a linked‐fragment approach or elongating the scaffold by means of combinatorial chemistry. Drug Dev. Res. 58:268–282, 2003. © 2003 Wiley‐Liss, Inc.

Early formation of a beta hairpin during folding of staphylococcal nuclease H124L as detected by pulsed hydrogen exchange.

Pulsed hydrogen exchange methods were used to follow the formation of structure during the refolding of acid-denatured staphylococcal nuclease containing a stabilizing Leu substitution at position 124 (H124L SNase). The protection of more than 60 backbone amide protons in uniformly (15)N-labeled H124L SNase was monitored as a function of refolding time by heteronuclear two-dimensional NMR spectroscopy. As found in previous studies of staphylococcal nuclease, partial protection was observed for a subset of amide protons even at the earliest folding time point (10 msec). Protection indicative of marginally stable hydrogen-bonded structure in an early folding intermediate was observed at over 30 amide positions located primarily in the beta-barrel and to a lesser degree in the alpha-helical domain of H124L SNase. To further characterize the folding intermediate, protection factors for individual amide sites were measured by varying the pH of the labeling pulse at a fixed refolding time of 16 msec. Protection factors >5.0 were observed only for amide positions in a beta-hairpin formed by strands 2 and 3 of the beta-barrel domain and a single site near the C-terminus. The results indicate that formation of stable hydrogen-bonded structure in a core region of the beta-sheet is among the earliest structural events in the folding of SNase and may serve as a nucleation site for further structure formation.

N-O glucuronidation: a major human metabolic pathway in the elimination of two novel anticonvulsant drug candidates

1. The urinary metabolites of the anti-convulsant compound 4-amino-1-(2,6-difluorobenzyl)-1H-1,2,3-triazolo[4,5-c]-pyridine hydrochloride (GI265080) obtained following a single oral dose to man have been detected and quantified relative to each other using 19F-NMR spectroscopy. 2. The human urinary metabolites of GI265080 were isolated using semipreparative HPLC and unequivocally characterized using 1H-NMR spectroscopy, two-dimensional heteronuclear NMR spectroscopy and mass spectrometry. The assignments of the N-(5)oxide and the N-(5)-O-glucuronide metabolites of GI265080 were further confirmed by independent synthesis. The urinary metabolites obtained following single oral doses to dog and rat have also been isolated and characterized. 3. The human urinary metabolites of GI265080 comprise the N-(5)-oxide, the quaternary N+-(5)-glucuronide, the 7-hydroxy glucuronide and a glucuronide conjugate of the N-(5)-oxide. The N-(5)-O-glucuronide conjugate is a novel species in human metabolism and is a significant route of elimination of GI265080 in man. 4. The urinary metabolites of the potential anti-convulsant GW273293 (6-amino-3-(2,3,5-trichlorophenyl)pyrazin-2-ylamine) obtained following a single oral dose to man have also been isolated and characterized. The formation of a novel N-O-glucuronide was also observed and was shown to constitute a significant route of elimination of GW273293 in man.

Characterization of the folding and unfolding reactions of a small β-barrel protein of novel topology, the MTCP1 oncogene product P13

The equilibrium and kinetic folding properties of a small oncogene product, P13MTCP1, of novel topology have been investigated using perturbation by guanidine hydrochloride and observation by fluorescence, circular dichroism and two-dimensional heteronuclear NMR spectroscopy. The structure of P13MTCP1 is comprised of a canonical filled β-barrel, although the topology of the structure is absolutely unique, rendering the folding properties of this protein of great interest. Equilibrium measurements of the intrinsic fluorescence emission spectrum, the fluorescence decay, the circular dichroism spectrum and the 15N-1H heteronuclear single quantum coherence (HSQC) correlation spectrum as a function of increasing concentrations of denaturant showed no evidence for the population of any equilibrium intermediates, although negative amplitudes on the blue edge of the tryptophan emission and loss of intensity of the native HSQC correlation peaks were indicative of increased conformational dynamics at low denaturant concentrations. The free energy and cooperativity of unfolding as observed by fluorescence and circular dichroism were in relatively good agreement, also consistent with a two-state transition. Kinetics measurements of the fluorescence emission as a function of denaturant concentration revealed that P13MTCP1 is the slowest folding β-structure protein reported to date. Comparison of the activation cooperativity values (mf and mu) indicates that the structure of the transition state is quite close to the folded state in terms of exposed surface area. The calculated contact order of P13MTCP1 is relatively low and does not appear to explain its slow rate of folding. We suggest that the complex topology of this protein, which would require the ordering of the β-barrel through a long loop joining the two L-shaped components of the barrel, could provide an explanation for this slow folding.

Role for phosphatidylinositol in nuclear envelope formation

PtdIns is a minor membrane phospholipid that is important in signal transduction. Recently, derivatives of PtdIns phosphorylated at the 3-position of the inositol ring have been implicated in the regulation of constitutive membrane traffic and in membrane fusion events. Assembly of the nuclear envelope (NE), a crucial step in the progress of mitosis, is also likely to involve membrane fusion reactions. We therefore investigated the role of PtdIns and phosphoinositide 3-kinase (PI-3K) activity in NE formation in vitro. GTP-induced NE formation was blocked by wortmannin and LY294002, two specific inhibitors of PI-3K, suggesting a role for PtdIns phosphorylated at the 3-position. PtdIns-specific phospholipase C mimicked GTP hydrolysis as an inducer of NE formation. This induction was dependent on a membrane vesicle subfraction (MV1) that was highly enriched in PtdIns, as determined by heteronuclear two-dimensional NMR spectroscopy. On the basis of these results, we suggest that the MV1 population serves as a source of membranes rich in PtdIns that might facilitate fusion, possibly through the production of the membrane-destabilizing lipid diacylglycerol.

Role for phosphatidylinositol in nuclear envelope formation.

PtdIns is a minor membrane phospholipid that is important in signal transduction. Recently, derivatives of PtdIns phosphorylated at the 3-position of the inositol ring have been implicated in the regulation of constitutive membrane traffic and in membrane fusion events. Assembly of the nuclear envelope (NE), a crucial step in the progress of mitosis, is also likely to involve membrane fusion reactions. We therefore investigated the role of PtdIns and phosphoinositide 3-kinase (PI-3K) activity in NE formation in vitro. GTP-induced NE formation was blocked by wortmannin and LY294002, two specific inhibitors of PI-3K, suggesting a role for PtdIns phosphorylated at the 3-position. PtdIns-specific phospholipase C mimicked GTP hydrolysis as an inducer of NE formation. This induction was dependent on a membrane vesicle subfraction (MV1) that was highly enriched in PtdIns, as determined by heteronuclear two-dimensional NMR spectroscopy. On the basis of these results, we suggest that the MV1 population serves as a source of membranes rich in PtdIns that might facilitate fusion, possibly through the production of the membrane-destabilizing lipid diacylglycerol.

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for post-translational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight β-turn that houses the lysine residue targetted for biotinylation, and the β-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct post-translational modification.

Solution structure of ileal lipid binding protein in complex with glycocholate.

Ileal lipid binding protein (ILBP) is a cytosolic lipid-binding protein that binds both bile acids and fatty acids. We have determined the solution structure of porcine ILBP in complex with glycocholate by homonuclear and heteronuclear two-dimensional NMR spectroscopy. The conformation of the protein-ligand complex was determined by restrained energy minimization and simulated annealing calculations after docking the glycocholate ligand into the protein structure. The overall tertiary structure of ILBP is highly analogous to the three-dimensional structures of several other intracellular lipid binding proteins (LBPs). Like the apo-structure, the bile-acid complex of ILBP is composed of 10 anti-parallel beta-strands that form a water-filled clam-shell structure, and two short alpha-helices. Chemical shift data indicated that the bile acid ligand is bound inside the protein cavity. Furthermore, 13C-edited heteronuclear single-quantum correlation-NOESY experiments showed NOE contacts between several aromatic residues located in the proposed bile acid portal region and the 13C-labeled ligand. A single bile acid molecule is bound inside the protein, with the steroid moiety penetrating deep into the water-accessible internal cavity, such that ring A is located right above the plane of the Trp49 indole ring. The carboxylate tail of the ligand is protruding from the proposed bile acid portal into the surrounding aqueous solution. The body of the steroid moiety is oriented with the nonpolar face in contact with the mostly hydrophobic residues of beta-strands C, D and E, while the polar face shows contacts with the side-chains of Tyr97, His99, Glu110 and Arg121 in beta-strands H, I and J. Thus, the conformational arrangement of the ligand complex suggests that the binding affinity of ILBP for bile acid molecules is based mainly on strong hydrophobic interactions inside the protein cavity. Furthermore, this binding mode explains how ILBP can transport unconjugated and conjugated bile acids.

Characterization of a type of ?-bend ribbon spiral generated by the repeating (Xaa-Yaa-Aib-Pro) motif: The solution structure of harzianin HC IX, a 14-residue peptaibol forming voltage-dependent ion channels

The three-dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two-dimensional nmr spectroscopy combined with molecular modeling. This 14-residue peptide, Ac Aib1 Asn2 Leu3 Aib4 Pro5 Ala6 Ile7 Aib8 Pro9 Iva10 Leu11 Aib12 Pro13 Leuol14 (Aib, α-aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short-sequence peptaibol class characterized by an acetylated N-terminus, a C-terminal amino alcohol, and the presence of three Aib-L-Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20-residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane-perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage-gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence-specific 1H- and 13C-nmr assignments and conformational nmr parameters (3JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty-seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β-turns twisted into a continuous spiral from Asn2 to Leuol14 and forming a 26 A long helix-like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn2 side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φi = −90°, Ψi = −27°; Φi+1 = −98°, Ψi+1 = −17°; Φi+2 = −49°, Ψi+2 = −50°; Φi+3 = −78°, Ψi+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)n sequences, the (Xaa–Yaa–Aib–Pro)-β-bend ribbon spiral. It is stabilized by 4 1 intramolecular hydrogen bonds and differs from both the canonical 310-helix made of a succession of type III β-turns and from the β-bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999

Mapping of a protein-RNA kissing hairpin interface: Rom and Tar-Tar*.

An RNA 'kissing' complex is formed by the association of two hairpins via base pairing of their complementary loops. This sense-antisense RNA motif is used in the regulation of many cellular processes, including Escherichia coli ColE1 plasmid copy number. The RNA one modulator protein (Rom) acts as a co-regulator of ColE1 plasmid copy number by binding to the kissing hairpins and stabilizing their interaction. We have used heteronuclear two-dimensional NMR spectroscopy to map the interface between Rom and a kissing complex formed by the loop of the trans -activation response (Tar) element of immunodeficiency virus 1 (HIV-1) and its complement. The protein binding interface was obtained from changes in amide proton signals of uniformly 15N-labeled Rom with increasing concentrations of unlabeled Tar-Tar*. Similarly, the RNA-binding interface was obtained from changes in imino proton signals of uniformly 15N-labeled Tar with increasing concentrations of unlabeled Rom. Our results are in agreement with previous mutagenesis studies and provide additional information on Rom residues involved in RNA binding. The kissing hairpin interface with Rom leads to a model in which the protein contacts the minor groove of the loop-loop helix and, to a lesser extent, the major groove of the stems.

1H and13C NMR assignments of a lipopolysaccharide obtained from the deep rough mutant ofEscherichia coliD31m4

The hexamethyl derivative of lipopolysaccharide (LPS), obtained by diazomethane methylation of LPS extracted from a deep mutant of Escherichia coli D31m4, followed by high-performance liquid chromatographic purification, was subjected to homo- and heteronuclear two-dimensional NMR spectroscopy (DQF-COSY, HOHAHA, NOESY, HMQC and HMBC) to achieve 1H and 13C NMR assignments, particularly of the sugar backbone. These studies confirmed two each of glucosamine (GlcN), 3-deoxy-D-manno-octulosonic acid (Kdo), phosphate, β-hydroxymyristate and β-acyloxymyristate and the structure of the tetrasaccharide as α-KdoP-(2→4)-α-KdoP-(2→6)-β-D-GlcPN-(1→6)α-D-GlcPN, bisphosphorylated at the 1 and 4′ positions. © 1998 John Wiley & Sons Ltd.

Structure and conformation of the procyanidin B-2 dimer

The structure of the proanthocyanidin B-2 dimer was proved to be (—)epicatechin-(4β-8)-(—)epicatechin by two-dimensional heteronuclear NMR spectroscopy. The compound adopts two conformations in solution, in which the interflavan dihedral angles are roughly ±90°. The preferred conformation of the pyran ring is a half-chair with the 3′,4′-dihydroxyphenyl substituents in pseudo-equatorial positions. © 1997 John Wiley & Sons, Ltd.

Structure and conformation of the procyanidin B-2 dimer

The structure of the proanthocyanidin B-2 dimer was proved to be (—)epicatechin-(4β-8)-(—)epicatechin by two-dimensional heteronuclear NMR spectroscopy. The compound adopts two conformations in solution, in which the interflavan dihedral angles are roughly ±90°. The preferred conformation of the pyran ring is a half-chair with the 3′,4′-dihydroxyphenyl substituents in pseudo-equatorial positions. © 1997 John Wiley & Sons, Ltd.

Backbone dynamics of proteins studied by two-dimensional heteronuclear NMR spectroscopy and molecular dynamics simulations

Backbone dynamics of proteins studied by two-dimensional heteronuclear NMR spectroscopy and molecular dynamics simulations