Amide Proton Resonances Research Articles

A powerful method of sequential proton resonance assignment in proteins using relayed 15N- 1H multiple quantum coherence spectroscopy

A powerful method of sequential resonance assignment of protein 1H-NMR spectra is presented and illustrated with respect to the DNA-binding protein ner from phage Mu. It is based on correlating proton-proton through-space and through-bond connectivities with the chemical shift of the directly bonded 15N atom. By this means, ambiguities arising from chemical shift degeneracy of amide proton resonances can be resolved. The experiments described involve combining the 1H-detected heteronuclear multiple quantum coherence correlation experiment with homonuclear nuclear Overhauser enhancement, J-correlated or Hartmann-Hahn experiments.

Line narrowing of amide proton resonances in 2D NMR spectra of proteins

(14) (a) The rates of alkyl radical addition to mono-substituted alkenes depend on the nature of the alkene substituents. For example, the addition rates to CH2=CHCOCH, are normally double those to CHI=CHCO2CH3 (ref 14b). Importantly, simple a-amino alkyl radicals are electron rich (high-energy SOMO) and thus should add to electron poor olefins more efficiently. This should be contrasted to the chemistry of a-amido alkyl radicals which add intramolecularly to simple alkene groupings (ref 14c). (b) Giese, B. Angew. Chem., In t . Ed. Engl. 1983, 764, 763 and references therein. (c) Hart, D. J . ; Tasi, Y . M. J . Am. Chem. SOC. 1982, 104, 1430. Burnett, D. A.; Choi, J . K.; Hart, D. J.; Tsai, Y . M. Ibid. 1984, 106, 8201. (1 5 ) For example, the comparative lack of and modest stereoselectivity of the respective direct and SET-sensitized photocyclizations of 9 needs to be addressed. Line Narrowing of Amide Proton Resonances in 2D NMR Spectra of Proteins

Folding of immunogenic peptide fragments of proteins in water solution: I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of β-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (β) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the β-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the β-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all l-amino acids except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the β-turn population. The β-turn populations in water solution measured in this way correlate with the β-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the β-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3 J N α coupling constants. The predominant structure appears to be a type VI β-turn, with cis-proline at position 3. Estimates of the β-turn population suggest that, in the most favourable peptides, as much as 70% of the cis isomer may exist in type VI turn conformations. The turn population for the trans peptides is smaller, but may be as high as 50% for certain peptides. The observed stability of β-turns in small peptide fragments of proteins suggests that turns are to be expected in polypeptide chains under folding conditions where they may have a profound influence on protein folding pathways.

Conformational Energy Calculations on the Eosinophil Chemotactic Peptide Analog Val-Gly-Ala-Glu: Comparison of Theory and Experiment

Conformational energy calculations have been employed to obtain minimum energy conformations of the peptide Val- Gly-Ala-Glu , an analogue of eosinophil chemotactic tetrapeptides. The calculated conformations of the peptide can be described as an ensemble of structures in which the C-terminal and N-terminal regions of the molecule are in close proximity. The charge state of the peptide showed a marked effect on the calculated conformation, and the results were also sensitive to the electrostatic environment. The calculations performed on the dianionic form of the molecule showed good agreement with experimental n.m.r . Data on coupling constants, amide-proton resonance chemical shifts and temperature coefficients, nuclear Overhauser effects, side-chain rotamer populations, and binding of paramagnetic ions, obtained in dimethyl sulfoxide solutions. The calculations demonstrate some of the inherent problems facing theoretical calculations of peptide structure.

Sequence-specific assignments of downfield-shifted amide proton resonances of calmodulin Use of two-dimensional NMR analysis of its tryptic fragments

Two-dimensional NMR methods were applied to assign the extremely downfield-shifted amide-proton resonances in the 500-MHz 1H-NMR spectra of the NH 2-terminal fragment of residues 1–75 of calmodulin. The low-field resonances of the 1H-NMR spectra of intact calmodulin were assigned to specific amino acid residues by comparison with spectra of the tryptic fragments of residues 1–75 and 78–148, in both the Ca 2+-free and Ca 2+-bound states. The hydrogen bonding of glycine residues connecting the two amino acid residues at the Z and − Y positions in the octahedral Ca 2+ coordination site was investigated. The Gly 134 in site IV showed a different property from the other glycines, 25, 61 and 98, involved in sites I, II and III, respectively.

NMR of a synthetic peptide spanning the triphosphate binding site of adenosine 5'-triphosphate in actin.

The amino acid residues 114-118 in actin were found to be implicated strongly in the binding of nucleotide, and as would be expected for such an important binding site, they are located in a completely conserved region of the actin sequence. A 19-residue peptide with the actin sequence 106-124 was synthesized in order to span the putative triphosphate binding site. Proton NMR spectra of the actin peptide 114-118 in the presence and absence of ATP indicated that Arg-116 and Lys-118 are particularly involved in binding ATP. A strong binding of ATP to the peptide 106-124 also was measured. Tripolyphosphate bound to the peptide 106-124 somewhat more weakly than ATP. Binding involved residues 115-118 and 121-124, indicating the presence of a reverse turn between these segments. Proton resonances were assigned by using two-dimensional double quantum correlated spectroscopy, one-dimensional spin decoupling techniques, one-dimensional nuclear Overhauser enhancement difference spectroscopy, and pH titration. The alpha CH resonances of Ala-3 and Asn-6 are markedly shifted downfield with respect to values in small unstructured peptides due to their close proximity to the side chains of Pro-4 and Pro-7, respectively. Several other resonances display chemical shifts which are indicative of a structured environment. Assignment of the amide proton resonances in H2O and measurements of the coupling constant 3JHNCH and the chemical shifts of the amide protons reveal that much of the synthetic peptide, particularly the backbone, exhibits a highly structured environment and represents a good model for the triphosphate binding site in actin.

Conformation of the glycotripeptide repeating unit of antifreeze glycoprotein of polar fish as determined from the fully assigned proton n.m.r. spectrum

With its simple glycotripeptide repeating structure the antifreeze glycoprotein of polar fish may be an especially simple conformational mode for mucin glycoproteins with similar but more complex structures. The fully assigned proton n.m.r. spectrum confirms the anomeric configurations of the hexapyranosidic sugars of the side chains and the coupling constants of the alpha GalNAc and the beta Gal residues show both to be in the expected 4C1 chair conformation. The assignment of a single resonance for each proton of the (Ala-Thr-Ala)n repeat unit coupled with the observation of long range nuclear Overhauser effects (n.O.e.) implies a three-fold repeating conformation. The resonances of the two alanines are distinct and can be assigned to their correct positions in the peptide sequence by n.O.e. observed at the amide proton resonances on saturation of the alpha proton signals. The amide proton coupling constants of all three peptide residues are similar and imply a limited range of peptide backbone torsion angles, phi CN. The large n.O.e. which has been observed between the amide proton and the alpha proton of the residue preceding it in the sequence implies large positive values for the peptide dihedral angle, psi CC. Limits are placed on possible values of side chain dihedral angles by the observation of the coupling constant between the alpha and beta protons of the threonyl residue. The observation of n.O.e. between the anomeric proton of GalNAc and the threonyl side chain protons gives information on the conformation of the alpha glycosidic linkage between the disaccharide and the peptide. n.O.e. observed between the protons of the beta glycosidic linkage indicates the conformation of the disaccharide and the large amide proton coupling constant of the GalNAc residue shows a trans proton relationship. The spectroscopically derived data have been combined with conformational energy calculations to give a conformational model for antifreeze glycoprotein in which the hydrophobic surfaces of the disaccharide side chains are wrapped closely against a three-fold left handed helical peptide backbone. The hydrophilic sides of the disaccharides are aligned so that they may bind to the ice crystal face, which is perpendicular to the fast growth axis inhibiting normal crystal growth.

Hydrogen exchange of individual amide protons in the F helix of cyanometmyoglobin.

Hydrogen exchange of the individual amide protons of alanine-90 (F5), glutamine-91 (F6), serine-92 (F7), and histidine-93 (F8) residues in cyanometmyoglobin of sperm whale has been studied by 1H nuclear magnetic resonance spectroscopy at 360 MHz. The amide proton resonance of F5, F6, and F7 have been assigned by use of the selective nuclear Overhauser effect between the consecutive amide protons. At pH 6.8, and in the temperature range of 5-20 degrees C, these protons show a 10(4)-fold retardation compared to the rates in free peptides. Apparent activation enthalpies for hydrogen exchange of F5, F6, and F8 protons are 18.5 +/- 0.4, 9.5 +/- 0.3, and 18.5 +/- 0.3 kcal/mol, respectively. Some implications of these results on the nature of the opening processes involved in hydrogen exchange are considered.

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.

Amide 1H n.m.r. study of the folding of ribonuclease C-peptide

The folding of ribonuclease A 1–13 (C-peptide) in H 2O near 0°C has been monitored by means of the amide and side chain NH proton resonances. The C-peptide carboxylate at low temperature forms, in a significant amount, a folded structure similar to the one that the 1–19 S-peptide adopts in the same conditions (3–13 α-helix). A quantitative comparison between helix stabilities of the lactone and carboxylate forms of C-peptide and S-peptide is reported. It is concluded that the proposed His 12 + … Hse 13 (COO − salt bridge, which competes with the one-turn stabilizing salt bridge His 12 + … Glu 9 − in the C-peptide carboxylate, does not suppress helix formation as previously suggested but it merely reduces its stability. The behaviour of the N 5-H resonance of the Arg 10 + side chain provides evidence for its implication in a further stabilizing interaction, most probably with Glu 2 −.

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme.

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: The amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H 2O at pH 2.5 or 5.5 were examined by 1H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-β- d-glucopyranose 3 3 Abbreviations used: HA, hyaluronic acid; GlcNAc, 2-acetamido-2-deoxy-β- d-glucopyranose; GlcUA, β- d-glucopyranuronic acid; IdUA, α- l-idopyranuronic acid; Me β-GlcNAc, methyl 2-acetamido-2-deoxy-β- d-glucose; TMS, trimethylsilane. (β-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to d-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the β-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.

Nuclear-magnetic-resonance studies on the conformations of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution. Conformation-activity relationship.

The conformation of tridecapeptide alpha-mating factor from yeast Saccharomyces cerevisiae in aqueous solution was analyzed, in comparison with those of active analog and inactive analog peptides. 270-MHz 1H-NMR spectra of these peptides were observed and the spectral patterns of main-chain N-H proton resonances were classified into three groups. alpha-mating factor and Trp1-bearing active peptides belong to the group A1, active des-Trp1-peptides belong to the group A2 while the peptides of group B are inactive. The main-chain N-H proton resonances of the groups A1 and A2 and side-chain N-H proton resonances were all assigned to individual residues. The 13C-NMR analysis of alpha-mating factor indicates that the Lys7-Pro8 and Gln10-Pro11 peptide bonds exclusively take the trans form. From the temperature and pH dependences of chemical shifts and Gd(III)-induced relaxation enhancements of amide proton resonances, alpha-mating factor is found to take partly a folded conformation in aqueous solution, with an alpha-helical form in the N-terminal domain and two beta-turn forms in the central and C-terminal domain. The pH dependence of fluorescence intensity indicates that, in this folded conformation, the C-terminal carboxylate group lies close to the N-terminal domain. The presence of the folded form in the N-terminal domain and the beta-turn form in the central domain correlates with the biological activity of alpha-mating factor and analog peptides. However, the folded conformation of alpha-mating factor is in equilibrium with predominantly unordered form, as found from the circular dichroism and NMR analyses. The N-H proton and C-alpha proton resonances of free alpha-mating factor as assigned in the present study allow the transferred nuclear Overhauser enhancement (NOE) analysis of the membrane-bound conformation that is more directly related with the activity.

Synthetic peptide analogs of skeletal troponin C: Fluorescence studies of analogs of the low-affinity calcium-binding site II

Two 12-residue peptides were synthesized by the solid-phase method as structural analogs of a Ca 2+-binding loop of rabbit skeletal troponin C. The sequence of the analogs corresponds to the binding loop of the Ca 2+-specific low affinity binding site II (residues 63–74) but with two amino acid substitutions. In one analog, Phe-72 was replaced by tyrosine. In the other Gly-66 was substituted by serine and Phe-72 by tyrosine. The intrinsic fluorescence of the peptides was enhanced upon addition of Tb 3+ or large excess of Ca 2+. From the enhancement of Tb 3+ emission association constants in the range (2–3) × 10 5 m −1 and a binding stoichiometry of 1 were determined for Tb 3+ binding to the peptides. Large excess of Ca 2+ displaced Tb 3+ from the Tb 3+-peptide complexes and from these results apparent stability constants of 500–700 m −1 were deduced for Ca 2+ binding. Preliminary proton nuclear magnetic resonance results on one of the peptides indicated that La 3+ induced considerable perturbation of the amide proton resonances of several residues, including the aspartate at position 3, the tyrosine at position 10, and the two glutamates at the C-terminus. The results suggest involvement of these residues in cation coordination.

Multiple Conformations and Conformational Change in the Acetonitrile of Cation-binding Cyclic Tetrapeptide, Cyclo[Gly–l-Cys(Bzl(OMe))–Sar–l-Pro

Abstract The conformations of the cyclic tetrapeptide, cyclo[Gly–l-Cys(Bzl(OMe))–Sar–l-Pro] (CGCSP), in solution, were studied by means of 1H-, 13C-nuclear magnetic resonance (NMR), and circular dichroism (CD). The CGCSP equilibrates among three conformers, M, m, and n, in acetonitrile, providing three sets of resonances for each proton in the 1H-NMR spectrum. An inspection of a CPK model indicated that the three structures were sterically allowed to occur by the cis-trans isomerism around the Cys-Sar and Sar-Pro peptide bond. The temperature dependence of the amide proton resonances in 1H-NMR suggested that one γ-turn (1←3 hydrogen bond from the Cys to the Pro) is present in the two conformers, M and m. Thus, the three conformations in acetonitrile were deduced from all of the spectral data. This cyclic tetrapeptide is capable of binding a monovalent cation, Li+, and divalent cations, Mg2+, Ca2+, and Ba2+. Its CD titration curves demonstrated that at least three types of complexes with Ca2+ and Ba2+ were formed. In contrast, Li+ and Mg2+ were shown to form the 1 : 1 complex with this pep tide. The NMR data suggest that the conformational change of the cyclic backbone takes place by changing the geometry of the peptide bonds around the Cys–Sar–Pro sequence on the addition of a metal cation.

A competing salt-bridge suppresses helix formation by the isolated C-peptide carboxylate of ribonuclease A

The C-peptide of ribonuclease A (residues 1 to 13) is obtained by cyanogen bromide cleavage at Met13, which converts methionine to a mixture of homoserine lactone (giving C-peptide lactone) and homoserine carboxylate (giving C-peptide carboxylate). The helix-forming properties of C-peptide lactone have been reported. The helix is formed intramolecularly in aqueous solution, is stabilized at low temperatures (0 to 20 °C) and also by a pH-dependent interaction between sidechains. The C-peptide lactone helix is about 1000-fold more stable than expected from “host-guest” data for helix formation in synthetic polypeptides. Here we report the failure of C-peptide carboxylate to form an α-helix in comparable conditions. Formation of a salt-bridge between the α-COO − group and the imidazolium ring of His12 + appears to be responsible for the suppression of helix formation. The presence of the Hse13-COO − … His12 + salt-bridge in C-peptide carboxylate is shown by 1H nuclear magnetic resonance titration of the amide proton resonances of His12 and Hse13, and is expected from model peptide studies. The most probable reason why C-peptide carboxylate does not form an α-helix is that the Hse13-COO − … His12 + salt-bridge competes successfully with a helix stabilizing salt-bridge (Glu9 − … His12 +). S-peptide (residues 1 to 20 of ribonuclease A) does form an α-helix with properties similar to those of the C-peptide (lactone) helix, which shows that the lactone ring of C-peptide lactone is not needed for helix formation. These results support the hypothesis that a Glu9 − … His12 + salt-bridge stabilizes the C-peptide (lactone) helix, and they show that specific interactions between side-chains can be important in preventing as well as in promoting α-helix formation.

Amide proton exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution: Studies with two-dimensional nuclear magnetic resonance

A novel approach for studies of amide proton exchange in proteins is presented. It relies on measurements of the amide proton-C α proton cross-peak intensities in the two-dimensional homonuclear correlated 1H nuclear magnetic resonance spectra. The protein is dissolved in 2H 2O and the solution is exposed to the conditions of p 2H and temperature where the exchange rates are to be measured. After variable intervals, the amide proton exchange in a sample of this protein solution is quenched by lowering the temperature and possibly by p 2H variation, and a COSY † † Abbreviations used: n.m.r., nuclear magnetic resonance; 2D n.m.r., 2-dimensional n.m.r.; COSY, 2D correlated spectroseopy; BPTI, basic pancreatic trypsin inhibitor; p.p.m., parts per million. spectrum of this sample is then recorded. Comparison of the NHC αH cross-peak intensities in the spectra recorded after different exchange times yields exchange rates for the individual amide protons. The main advantage compared to previously described techniques is that a much more complete set of individual amide proton exchange rates can be obtained. In the basic pancreatic trypsin inhibitor, where all the amide proton resonances were previously individually assigned, quantitative exchange rates were obtained for 38 of the total of 53 backbone amide protons, and for 14 additional protons lower limits for the exchange rates were established from comparison of the COSY spectra recorded in H 2O and in 2H 2O. Proton exchange data were thus for the first time obtained for numerous peptide groups that are located near the protein surface in the single crystal structure of BPTI. For some locations on the protein surface, it appears that the amide proton exchange rates cannot be correlated readily with the static accessible surface areas in the crystal structure.

Nuclear magnetic resonance analysis of Gd 3+-induced perturbations in thymopoietin 32–36: A study of amide and aromatic proton resonances

The Gd 3+-induced perturbations in the NMR spectra of a cell differentiating peptide fragment, ArgLysAspValTyr (TP5), have been examined. This pentapeptide fragment retains the selective T-cell differentiating activity of its parent polypeptide thymic hormone, thymopoietin. The observed relaxation enhancements induced by Gd 3+ have been analyzed to determine the relative and absolute amide and aromatic proton-Gd 3+ distances. The data are compatible with a bidentate model, in which both the aspartyl and tyrosyl carboxylates bind the metal ion simultaneously in a chelate fashion, being the dominant conformer. From these studies a picture of the conformation of Ln 3+ complexes of TP5 begins to emerge.

Individual assignments of the amide proton resonances involved in the triple-stranded antiparallel pleated beta-sheet structure of a long neurotoxin, Laticauda semifasciata III from Laticauda semifasciata.

THe characteristic feature of the crystal structure of erabutoxin b, a short neurotoxin from Laticauda semifasciata, and alpha-cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple-stranded antiparallel pleated beta-sheet structure formed by the central and the third peptide loops. In the present study, we have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L. semifasciata, using nuclear Overhauser effects (NOE) and spin-decoupling methods. The results show that nearly all of the slowly exchangeable amide protons are to be assigned to the back-bone amide protons, involved in the triple-stranded antiparallel pleated beta-sheet structure, indicating that this sheet is stable in 2H2O solution. In contrast, the amide protons in short neurotoxins are readily exchangeable under the same experimental condition, suggesting that long neurotoxins have a more rigid sheet structure than short ones. This rigidity may come from the hydrophobic and hydrogen bond interaction between the central loop and the tail, which is not present in short neurotoxins. Since the functionally important residues are located on this beta-sheet, the different kinetic properties of the neurotoxins are well correlated with the difference in the rigidity of the beta-sheet.

Nuclear-magnetic-resonance study on Met-enkephalin and Met-enkephalinamide. Molecular association and conformation.

The 270-MHz 1H and 68-MHz 13C nuclear magnetic resonance spectra of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) and Met-enkephalinamide are analyzed in a variety of solvents. For the dipolar form of Met-enkephalin in (C2H3)2SO solution, significant concentration dependences are found of C-alpha proton chemical shifts, indicating an aromatic ring-current effect in molecular aggregates. An anomalous temperature dependence is observed of the amide proton chemical shift of the Met-5 residue. Furthermore, the chemical shifts of C-alpha protons of the dipolar form are found to depend appreciably on temperature. From the analyses of the temperature dependences together with concentration dependences of C-alpha proton resonances, the dipolar form of Met-enkephalin is found to be in an equilibrium of folded and extended conformations at low concentration in (C2H3)2SO solution. Solvent-composition dependences of the amide and C-alpha proton resonances and carbonyl and alpha-carbon resonances of the dipolar form in 2H2O/(C2H3)2SO solution are consistent with the conformation equilibrium and the association equilibrium. The folded conformation of the dipolar form in (C2H3)2SO solution is stabilized by the intramolecular attraction between the positively charged N-terminal group and negatively charged C-terminal group. The presence of the folded conformation is confirmed by the measurements of Gd(III)-induced relaxation enhancements of C-alpha protons. Nuclear Overhauser effects on the dipolar form are not consistent with the predominant formation of the beta-turn structure with the intramolecular hydrogen bond (Gly-2) C=O . H-N(Met-5). For the dipolar form of Met-enkephalin in 2H2O solution and for the cationic form of Met-enkephalinamide in (C2H3)2SO solution and in 2H2O solution there is no evidence for the formation of folded conformations.