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.     

Nuclear magnetic resonance detection of bound water molecules in the active site of Lactobacillus casei dihydrofolate reductase in aqueous solution.

Proton nuclear magnetic resonance spectroscopy has been used to detect two water molecules bound to residues in the active site of the Lactobacillus casei dihydrofolate reductase (DHFR). Their presence was detected by measuring nuclear Overhauser effects between NH protons in protein residues and protons in the individual bound water molecules in two-dimensional nuclear Overhauser effect spectroscopy (NOESY), in nuclear Overhauser effect spectroscopy in the rotating frame (ROESY) and three-dimensional 1H-15N ROESY-heteronuclear multiple quantum coherence spectra recorded on samples containing appropriately 15N-labelled DHFR. For the DHFR-methotrexate-NADPH complex, two bound molecules were found, one close to the Trp5 amide NH proton and the other near to the Trp21 indole HE1 proton: these correspond to two of the water molecules (Wat201 and Wat253) detected in the crystal structure studies described by Bolin and co-workers. However, the nuclear magnetic resonance experiments did not detect any of the other bound water molecules observed in the X-ray studies. The nuclear magnetic resonance results indicate that the two bound water molecules that were detected have lifetimes in the solution state that are longer than approximately two nanoseconds. This is of considerable interest, since one of these water molecules (Wat253) has been implicated as the likely proton donor in the catalytic reduction of dihydrofolate to tetrahydrofolate.     

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.     

NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to make resonance assignments of the proton spectra of two defensin antimicrobial peptides, human neutrophil peptide HNP-1 and rabbit neutrophil peptide NP-2. The secondary structures of these peptides were determined from analysis of the proton-proton NOEs and from the positions of slowly exchanging amide protons. Both peptides contain a long stretch of a double-stranded antiparallel beta-sheet in a hairpin conformation that contains a beta-bulge, a short region of triple-stranded beta-sheet, and several tight turns. The NMR results clearly show that HNP-1 forms a dimer or higher order aggregate in solution and that Pro8 exists as a cis peptide bond. The NMR data on these peptides are compared with NMR data for a homologous peptide NP-5 [Bach, A. C., Selsted, M. E., & Pardi, A. (1987) Biochemistry 26, 4389-4397]. Analysis of the conformation-dependent proton chemical shifts shows that it is not possible to confidently judge the structural similarity of the three defensins from chemical shift data alone. However, comparison of the 3JHN alpha coupling constants in NP-2 and NP-5 indicates that the backbone conformations for these peptides are very similar. A more detailed comparison of the solution conformations of the defensins peptides is made in the following paper in this issue where the NMR data are used as input for distance geometry and molecular dynamics calculations to determine the three-dimensional structures of HNP-1 and NP-2.     

Sequence-specific recognition of DNA: assignment of nonexchangeable proton resonances in the consensus Pribnow promoter DNA sequence by two-dimensional NMR

The resonances of most of the nonexchangeable protons of both + and - strands of the consensus Pribnow dodecamer d( CGTTATAATGCG ) have been assigned by two-dimensional nuclear magnetic resonance methods. Application of the two-dimensional nuclear Overhauser effect ( NOESY ) sequential connectivity method, combined with two-dimensional autocorrelated ( COSY ) spectra to reveal scalar-coupled protons, results in assignment of virtually all of the base and sugar protons, except the sugar C5 protons which are inadequately resolved. Analysis of the nuclear Overhauser data indicates that the helix assumes a fairly uniform B form conformation.     

Sequence-specific 1H NMR assignments and identification of slowly exchanging amide protons in murine epidermal growth factor

Proton nuclear magnetic resonance (1H NMR) assignments for the murine epidermal growth factor (mEGF) in aqueous solution were determined by using two-dimensional NMR at pH 3.1 and 28 degrees C. The assignments are complete for all backbone hydrogen atoms, with the exception of the N-terminal amino group, and for 46 of the 53 side chains. Among the additional seven amino acid residues, three have complete assignments for all but one side-chain proton, and between two and four protons are missing for the remaining four residues. The sequential assignments by nuclear Overhauser effect spectroscopy are consistent with the chemically determined amino acid sequence. The NMR data show that the conformations of both the Tyr3-Pro4 and Cys6-Pro7 peptide bonds are trans in the predominant solution structure. Proton-deuterium exchange rate constants were also measured for 13 slowly exchanging amide protons. The information presented here has been used elsewhere to determine the three-dimensional structure of mEGF in aqueous solution.     

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.     

Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

Conformation of a neurokinin antagonist in solution. 2D NMR and restrained molecular dynamics study.

The hexapeptide [cyclo(Leu1 psi(CH2NH2)Leu2-Gln3-Trp4-Phe5-Gly6)]+1 is a potent antagonist of neurokinin A activity in tissues of hamster urinary bladder. The solution conformation of this cyclic hexapeptide has been characterized by the combined use of two dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton spectrum of the peptide was fully assigned by the sequential assignment procedure. Interproton distances were derived from crosspeak volumes in two dimensional Nuclear Overhauser Effect spectra, and dihedral angles were calculated from appropriate coupling constants. Temperature coefficients of the amide protons were determined. Restrained molecular dynamics simulations were carried out using the backbone interproton distances as constraints. During 210 ps of restrained molecular dynamics the peptide interconverted among three closely related families of conformations. These interconversions occurred at picosecond timescales under the simulation conditions.     

Total assignments, including four aromatic residues, and sequence confirmation of the decapeptide tyrocidine A using difference double resonance. Qualitative nuclear overhauser effect criteria for beta turn and antiparallel beta-pleated sheet conformations.

The complete assignments of all the proton magnetic resonance signals from each NH-CalphaH-CbetaH2 moiety in a complex peptide containing several residues of the same type has not yet been achieved without specific or stereospecific isotopic enrichment. We report the sequencing and proton magnetic resonance spectral assignments, including those of 4 aromatic residues, of tyrocidine A, an analog of the decapeptide gramicidin S. Two complementary methods, proton-proton nuclear Overhauser enhancements and scalar decoupling, evaluated by two distinct forms of difference double resonance, were used. All chemical shifts, scalar coupling constants, and [1H:1H] nuclear Overhauser enhancements for the backbone protons are reported. The [1H:1H] nuclear Overhauser enhancements are consistent with tyrocidine A possessing a beta-I turn/beta-II' turn/antiparallel beta-pleated sheet conformation. In addition to the previously proposed nuclear Overhauser enhancement criteria for beta turns and antiparallel beta sheets, another criterion for identifying the antiparallel beta sheet is demonstrated; namely, the nuclear Overhauser enhancement between 2 CalphaH protons of the central resisdues, in this case the Phe7CalphaH and Orn2CalphaH.     

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.     

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance.

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described. A distance geometry/simulated annealing routine was used to generate well-defined backbone and side-chain structures. The species isolated is a right-handed intertwined double helix, with approximately 5.7 residues per turn. Unique values for helical dimensions are also specified.     

Individual assignments of amide proton resonances in the proton NMR spectrum of the basic pancreatic trypsin inhibitor.

Studies of proton-proton nuclear Overhauser effects were used to obtain individual assignments of 17 amide proton resonances in the 360 MHz proton nuclear magnetic resonance spectrum of the basic pancreatic trypsin inhibitor. First, optimizing the conditions for obtaining selective nuclear Overhauser effects in the presence of spin diffusion in macromolecules is discussed. Truncated driven nuclear Overhauser experiments were used to assing the amide proton resonances of the beta-sheet in the inhibitor. It is suggested that these techniques could serve quite generally to obtain individual resonance assignments in beta-sheet secondary structures of proteins. Combination of nuclear Overhauser studies with spin decoupling further resulted in individual assignments of the gamma-methyl resonances of the two isoleucines and numerous Calpha and Cbeta protons.     

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.     

Sequential resonance assignments as a basis for the determination of a three-dimensional structure of protein E-L30 of Escherichia coli

Nuclear Overhauser enhancement spectra of ribosomal protein E-L30 were searched for interresidual connectivities involving peptide bond amide protons in order to establish sequential neighbourships between amino acid residues. By comparing these data with the actual amino acid sequence of the protein, sequential resonance assignments became available for almost 90% for the amino acids in E-L30. With the aid of these assignments, some 30 nuclear Overhauser connectivities could be interpreted in terms of short interproton distances involving remote sites in the polypeptide chain. It turned out that these contacts between residues generated enough constraints to permit construction of a three-dimensional structure for the protein.     

Hydrogen bonding in the carboxyl-terminal half-fragment 78-148 of calmodulin as studied by two-dimensional nuclear magnetic resonance

The C-terminal half-fragment (residues 78-148) of scallop testis calmodulin was investigated by 500-MHz two-dimensional proton NMR in order to clarify the structure and the structural change accompanying Ca2+ binding. The sequential resonance assignment to individual amino acid residues was made in part (27 out of 71 residues) by a combination of correlated spectroscopy and nuclear Overhauser effect spectroscopy of a 90% H2O solution. In the Ca2+-bound state, resonances of backbone amide protons of Gly-98, Gly-134, Ile-100, Asn-137, and Val-136 appear at extremely low fields. These findings suggest that amide protons of these residues are hydrogen bonded. In the Ca2+-free state, the amide resonances of Ile-100 and Gly-134 disappear into the crowded normal shift region. This observation indicates that two hydrogen bonds of Ile-100 and Gly-134 are destroyed (or weakened) as Ca2+ ions are removed from two Ca2+-binding sites. Chemical shifts of amide and alpha-protons of residues located in the Ca2+-binding loop of domain III are similar to those of domain IV. These results suggest that the conformations of the two loops are very similar. The present results can be interpreted in terms of a structure predicted by Kretsinger [Kretsinger, R.H. (1980) Ann. N.Y. Acad. Sci. 356, 14].     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

Structure and conformational heterogeneity of the weak toxin from the cobra Naja kaouthia venom

Resonances in the two-dimensional 1H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthia for all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequentional assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthia venom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis-trans isomerization of the peptide bond Arg32-Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin-spin coupling of protons, and rates of exchange of amide protons by deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel beta-sheets, one of which consists of two strands (regions 1-5 and 13-17) and the other, of three strands (regions 23-28, 38-43, and 55-59).     

Determination of a high precision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY cross-peaks

The solution structure of a novel 69 residue proteinase inhibitor, Linum usitatissimum trypsin inhibitor (LUTI), was determined using a method based on computer aided assignment of nuclear Overhauser enhancement spectroscopy (NOESY) data. The approach applied uses the program NOAH/DYANA for automatic assignment of NOESY cross-peaks. Calculations were carried out using two unassigned NOESY peak lists and a set of determined dihedral angle restraints. In addition, hydrogen bonds involving amide protons were identified during calculations using geometrical criteria and values of HN temperature coefficients. Stereospecific assignment of beta-methylene protons was carried out using a standard procedure based on nuclear Overhauser enhancement intensities and 3J(alpha)(beta) coupling constants. Further stereospecific assignment of methylene protons and diastereotopic methyl groups were established upon structure-based method available in the program GLOMSA and chemical shift calculations. The applied algorithm allowed us to assign 1968 out of 2164 peaks (91%) derived from NOESY spectra recorded in H2O and 2H2O. The final experimental data input consisted of 1609 interproton distance restraints, 88 restraints for 44 hydrogen bonds, 63 torsion angle restraints and 32 stereospecifically assigned methylene proton pairs and methyl groups. The algorithm allowed the calculation of a high precision protein structure without the laborious manual assignment of NOESY cross-peaks. For the 20 best conformers selected out of 40 refined ones in the program CNS, the calculated average pairwise rmsd values for residues 3 to 69 were 0.38 A (backbone atoms) and 1.02 A (all heavy atoms). The three-dimensional LUTI structure consists of a mixed parallel and antiparallel beta-sheet, a single alpha-helix and shows the fold of the potato 1 family of proteinase inhibitors. Compared to known structures of the family, LUTI contains Arg and Trp residues at positions P6' and P8', respectively, instead of two Arg residues, involved in the proteinase binding loop stabilization. A consequence of the ArgTrp substitution at P8' is a slightly more compact conformation of the loop relative to the protein core.     

Assignment of the non-exchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods

A general method of assigning the non-exchangeable protons in the nuclear magnetic resonance spectra of small DNA molecules has been developed based upon two-dimensional autocorrelated (COSY) and nuclear Overhauser (NOESY) spectra in 2H2O solutions. Groups of protons in specific sugars or bases are identified by their scalar couplings (COSY), then connected spatially in a sequential fashion using the Overhauser effect (NOESY). The method appears to be generally applicable to moderate-sized DNA duplexes with structures close to B DNA. The self-complementary DNA sequence d(C-G-C-G-A-A-T-T-C-G-C-G) has been synthesized by the solid-phase phosphite triester technique and studied by this method. Analysis of the COSY spectrum and the NOESY spectrum leads to the unambiguous assignment of all protons in the molecule except the poorly resolved H5' and H5" resonances. The observed NOEs indicate qualitatively that, in solution, the d(C-G-C-G-A-A-T-T-C-G-C-G) helix is right-handed and close to the B DNA form with a structure similar to that determined by crystallography.