13C-Nmr studies of ACTH: Assignment of resonances and conformational features

The biologically active ACTH(1–32) and ACTH(1–24) and other shorter peptide segments of the native hormone ACTH(1–39) were studied in aqueous solution by ¹³C-nmr. In order to identify the ¹³C resonances—except those of the carbonyls—both high-field nmr spectroscopy measurements and substitution of residues with amino acids enriched to 85% in ¹³C were carried out. The main results are (1) the direct characterization of the cis–trans isomerism of proline 24 and its effects on the directly connected and sequentially neighboring residues and (2) findings that the conformational features agree with an α-helix type organization in the N-terminal part of the ACTH molecule which is responsible for the biological activity.     

A 13C-NMR study on the conformational and dynamical properties of a cereal seed storage protein,C-hordein,and its model peptides

We have recorded the ¹³C CP-MAS and DD-MAS nmr spectra of dry and hydrated barley storage protein, C-hordein, as a model for wheat S-poor prolamins, together with those of model synthetic peptides (Pro)₂(Gln)₆(I) and (Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln)₃(II) under dry or hydrated conditions. The spectral features of C-hordein as well as these peptides were appreciably different from each other depending on the extent of hydration, reflecting different domains that adopt different types of conformations as well as dynamics. In particular, considerable proportions of the peak intensities were lost in the CP-MAS spectra, and well-resolved ¹³C-nmr signals emerged in DD-MAS nmr spectra owing to acquisition of molecular motions by swelling. It was shown that local β-turn or (Pro)_n type II conformation is more preferable for individual Pro residues and β-sheet type conformation is dominant for individual Gln residues in the dry and hydrated systems. In addition, two types of Gln environments are originated in C-hordein that differ in their mobility. Further, ¹³C spin-lattice relaxation times (T₁'s) of C- hordein and peptide II were reduced by more than one order of magnitude by hydration, reflecting the presence of well-swollen molecular chains. In contrast, theT₁ values of peptide I upon hydration remained one third of those in the dry state. Carbon-resolved proton spin-lattice relaxation times in the rotating frame (T_1ρ's) were also decreased by about 50% upon hydration, although these parameters were less sensitive as compared to T₁ values. In addition, the ¹³C-nmr signals of the aromatic side chain of Phe residues disappeared on hydration owing to interference between the frequency of the acquired flip-flop motion and the proton decoupling frequency. This information gives a new insight into establishing the structural properties of the studied protein system. A model may be put forward for a gel-type structure in which the more rigid part of the system involves intermolecular hydrogen-bonded Gln side chains as well as some hydrophobic “pockets” involving Pro and Phe residues. The liquid-like domain is characterized by considerable backbone and side-chain motion as well as rapid ring-puckering motion in Pro residues. © 1997 John Wiley & Sons, Inc.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

Conformational effects on peptide aggregation in organic solvents: spectroscopic studies of two chemotactic tripeptide analogs

The aggregation behavior of the chemotactic peptide analogs, Formyl-Met-Leu-Phe-OMe ( 1 ) and Formyl-Met-Aib-Phe-OMe ( 2 ), has been studied in chloroform and dimethylsulfoxide over the concentration range of 0.2–110 mM by ¹H-nmr spectroscopy. Both peptides associate in CDCl₃ at concentrations ≥ 2 mM, while there is no evidence for aggregation in (CD₃)₂SO. Analog 1 adopts an extended conformation in both solvents favoring association to form β-sheet structures. A folded, γ-turn conformation involving a 3 → 1 hydrogen bond between Met CO and Phe NH is supported by ¹H-, ¹³C-nmr, and ir studies of analog 2 . The influence of backbone conformation on the ease of peptide aggregation is demonstrated by ir studies in CHCl₃ and CD studies in dioxane.     

13C-NMR chemical shifts and the conformations of rigid polypeptides

¹³C-nmr chemical shifts of backbone carbonyl and side-chain β-carbons in polypeptides provide structural information. Recent utilization of substituent effects on ¹³C-nmr chemical shifts (principally γ-effects) has permitted the rationalization of their sequence and conformation dependence when observed in linear, flexible polypeptides. In this report, we apply the γ-effect method to study the ¹³C-nmr chemical shifts observed in solution and in the solid state for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides, which are usually cyclic. As found previously for flexible, linear polypetides, the relative ¹³C-nmr chemical shifts observed for the backbone carbonyl and side-chain β-carbons in conformationally rigid polypeptides are predictable from knowledge of their peptide residue sequence (primary structure) and conformation (secondary structure) via the γ-effect method.     

Conformational interconversions in peptide β-turns: Discrimination between enantiomeric conformations by chiral perturbation

The crystal structure of the model tripeptide Boc-Aib-Gly-Leu-OMe ( 1 ) reveals two independent molecules in the asymmetric unit that adopt “enantiomeric” type I and type I′ β-turn conformations with the Aib and Gly residues occupying the corner (i + 1 and i + 2) positions. ¹³C cross polarization and magic angle sample spinning spectra in the solid state also support the coexistence of two conformational species. ¹³C-nmr in CDCl₃ establishes the presence of a single species or rapid exchange between conformations. 400 MHz ¹H-nmr provides evidence for conformational exchange involving a major and minor species, with β-turn conformations supported by the low solvent exposure of Leu(3) NH and the observation of N_iH ↔ N_i+1H nuclear Overhauser effects. CD bands in the region 190–230 nm are positive, supporting a major population of type I′ β-turns. The isomeric peptide, Boc-Gly-Leu-Aib-OMe ( 2 ), adopts an “open” type II′ β-turn conformation in crystals. Solid state and solution nmr support population of a single conformational species. Chiral perturbation introduced outside the folded region of peptides may provide a means of modulating screw sense in achiral sequences. © 1998 John Wiley & Sons, Inc. Biopoly 45: 191–202, 1998     

Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins

Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [¹H]¹³C and [¹H]¹⁵N heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [¹H]¹³C heteronuclear NOE in combination with ¹³C-R ₁ can yield a more accurate analysis of side chain motional parameters. The observation of significant [¹H]¹⁵N heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.     

Molecular dynamics simulation and nmr study of a blood group H trisaccharide

Molecular dynamics simulations in vacuum and solution have been carried out on 2′-α-L -fucosyllactitol, a model for blood group H in conjunction with two-dimensional nmr measurements on the same compound. Three independent starting conformations for the dynamics were chosen from low energy conformations obtained by a ?/ψ grid search. Nine 5 ns vacuum simulations of the trisaccharide were performed, employing three different ways to treat electrostatic interactions for each starting conformation: distance-dependent dielectric with ε = r, constant dielectric with ε = 1, or constant dielectric with ε = 80. In vacuum, transitions of ? and ψ for the α-L -Fuc-(1 → 2)-β-D -Gal element occur in a cooperative manner. The virtual distance obtained for H1 in fucose to H2 in galactose from nuclear Overhauser effect spectroscopy experiments agree with one of the conformations of the trisaccharide in one of the three 100 ps aqueous simulations (?/ψ ca. ?100°/150°), indicating this may be a dominant solution conformation. The rms fluctuations of the ?- and ψ-dihedral angles were ～ 10° for a conformational state, both in the vacuum and the aqueous simulations. For the simulations in vacuum, the agreement with experimental NOE data is reasonable when a constant dielectric of 1 is used (major conformers having ?/ψ ca. ?100°/150° and ?140°/100°), whereas the agreement was poor with a constant dielectric of 80. Translational diffusion coefficients calculated from the simulation of the oligosaccharides were 0.12–0.18 × 10^?5 cm²/s and from nmr measurements 0.27 × 10^?5 cm²/s. © 1994 John Wiley & Sons, Inc.     

Hemoglobin aggregation in oxygenated sickle cells studies by carbon-13 rotating frame spin-lattice relaxation in the presence of an off-resonance radiofrequency field

Evidence is presented which shows that hemoglobin S in sickle cells has a tendency to aggregate even in the oxygenated state. The basis for that conclusion is derived from ¹³C-nmr rotating-frame spin–lattice relaxation studies in the presence of an off-resonance radiofrequency field in which the carbonyl resonances of hemoglobins in erythrocytes are examined. The experiments indicate that the rotational correlation time of hemoglobin S in oxygenated sickle cells at 38°C is 130 nsec compared to a value of 95 nsec for hemoglobin A in normal erythrocytes at the same temperature and the same mean cell hemoglobin content.     

Synthesis of 2S-Dioxo Isosteres of Purine and Pyrimidine Nucleosides IV. Selective Glycosylation of 4-Amino-5H-Imidazo [4, 5-c]-1, 2, 6-Thiadiazine 2, 2-Dioxide

Abstract

Selective glycosylation of 4-amino-5H-imidazo [4, 5-c]-1, 2, 6-thiadiazine 2, 2-dioxide (1) through its 1-benzyl derivative (2) is described. The structures of the compounds are discussed on the basis of ¹H nmr 2D homonuclear chemical shift correlations, NOE difference spectroscopy and iterative analyses.     

Synthesis of delta(E)Phe-containing tripeptide via photoisomerization and its conformation in solution

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.     

Nmr study of poly(aspartic acid). I. α- and β-Peptide bonds in poly(aspartic acid) prepared by thermal polycondensation

The structure of poly(aspartic acid) prepared by thermal polycondensation has been studied by means of nmr spectroscopy. The analysis of the ¹³C-nmr spectra of the polymer at various pH values and comparison with the spectrum of poly(α-L -aspartic acid) revealed that the polymer contained aspartic acid linked in α- and β-peptide bonds. The mole fraction of the β-peptide bonds has been found to be 0.8 ± 0.1. The significance of the results for the evolutionary theory of S. W. Fox is mentioned.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

1H- and 13C-nmr studies on caffeine and its interaction with nucleic acids

The relative orientation of caffeine in stacks formed by self-association in aqueous solution has been evaluated by both ¹H- and ¹³C-nmr spectroscopy. The data, which were interpreted through the calculation of ring-current and atomic diamagnetic anisotropic effects of the caffeine molecule, suggest two caffeine bases may stack in a nearly orthogonal manner. The interactions between caffeine and adenylyl-3′,5′-adenosine, polyadenylic acid, and ribo-(A-A-G-C-U-U)₂ helix were also studied by nmr at different caffeine/base ratios and at varying temperatures. The results show that caffeine tends to stack on the top of the terminal purine bases or to insert (the single-stranded) or to intercalate (the double-stranded) between purine bases.     

Complete relaxation matrix refinement of NMR structures of proteins using analytically calculated dihedral angle derivatives of NOE intensities

Summary A new method for refining three-dimensional (3D) NMR structures of proteins is described, which takes account of the complete relaxation pathways. Derivatives of the NOE intensities with respect to the dihedral angles are analytically calculated, and efficiently evaluated with the use of a filter technique for identifying the dominant terms of these derivatives. This new method was implemented in the distance geometry program DIANA. As an initial test, we refined 30 rigid distorted helical structures, using a simulated data set of NOE distance constraints for a rigid standard -helix. The final root-mean-square deviations of the refined structures relative to the standard helix were less than 0.1 Å, and the R-factors dropped from values between 7% and 32% to values of less than 0.5% in all cases, which compares favorably with the results from distance geometry calculations. In particular, because spin diffusion was not explicitly considered in the evaluation of exact¹H–¹H distances corresponding to the simulated NOE intensities, a group of nearly identical distance geometry structures was obtained which had about 0.5 Å root-mean-square deviation from the standard -helix. Further test calculations using an experimental NOE data set recorded for the protein trypsin inhibitor K showed that the complete relaxation matrix refinement procedure in the DIANA program is functional also with systems of practical interest.Abbreviations RMSD root-mean-square deviation - NOE nuclear Overhauser enhancement - NOESY 2-dimensional nuclear Overhauser enhancement spectroscopy - CPU central processing unit     

Secondary structure of calf-thymus histone H1 by means of 13C-NMR spectroscopy

¹³C-nmr spectroscopy was used to determine the location of the structured segment of the chain of histone H1 in water and NaCl solutions. The segment found (41–89) is contained in the area proposed by others on the basis of ¹H-nmr studies. The number of basic groups left in the mobile ends of the protein matches the number of bases of internucleosomal DNA.     

Study of the rotational diffusion properties of peptides in solution

In this paper we examine the theory and method for obtaining rotational diffusion coefficients for peptides in dilute solution from ¹³C-nmr spin-lattice relaxation data. We show that even for the case of nearly equal observed relaxation times of chemically and magnetically nonequivalent alpha-carbons marked rotational anisotropy will be the usual case. We describe two interactive, minicomputer programs which are of general use in this type of work. The implications of this study on spectral density-based conformational determinations of peptides is discussed.     

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance C NMR spectroscopy

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance ¹³C NMR spectroscopy that provides complementary information to 2D ¹H NMR. The correlation of ¹³Cα secondary shifts with both 3D structure and heteronuclear ¹⁵N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D ¹H-¹³Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. ¹H-¹³Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide-membrane interactions from different angles.     

Interaction of heparin with a synthetic pentadecapeptide from the C-terminal heparin-binding domain of fibronectin

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.     

Nmr studies on the presence of domain structures in polysaccharides

A variety of nmr transient techniques demonstrate the absence of domain structures in powders of α- and β-cyclodextrins, dextran B512F, and their “deuterated” analogs where deuterium replaces exchangeable protons. Cyclodextrins and dextran are inferred to be homogeneously ordered and disordered one-phase systems, respectively. We show how erroneous results concerning the presence and sizes of rigid and mobile domains can be reached from the interpretations of two-component free induction decays and spin–lattice relaxation behavior of polysaccharides. In our approach, we have measured ¹H free induction decays and lineshapes, and ¹H spin–lattice and dipolar relaxation times, in addition to using Goldman–Shen experiments and ¹³C cross-polarization magic-angel sample spinning. Self-consistent nmr results are suggested as essential means to corroborate conclusions concerning domain structures in biopolymers.