Characterization of the proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance. Stability, amide proton exchange and mobility of aromatic residues

The isoinhibitor IIA from bull seminal plasma was investigated in aqueous solution by 1H nuclear magnetic resonance (n.m.r.). The analysis of the 1H n.m.r. data was based on individual resonance assignments, which are described in the following paper. Large conformation-dependent chemical shifts for aliphatic amino acid side-chains, numerous slowly exchanging amide protons and unusual pH titrations of two aromatic residues show that this protein forms a compact, globular conformation. This form of the protein is stable between pH 4 and 12 at 25 degrees C, and between 5 and 50 degrees C at pH 4.9. At temperatures above 50 degrees C there is evidence for an equilibrium between several different conformations, with the rate of exchange between the different species being in the intermediate range on the n.m.r. time-scale. Preliminary data are presented for the individual exchange rates of 18 backbone amide protons. Among the four aromatic rings, Phe10, Phe38 and Tyr16 undergo rapid 180 flips over the entire temperature range, whereas for Tyr32 a temperature-dependent transition from low-frequency to high-frequency flipping motions was observed.     

Calcium and praseodymium complexes in solution. 1H n.m.r. conformational study of the model tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine

The synthetic tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine (Ac-DVDA) is a model of the calcium binding site of proteins such as carp parvalbumin, thermolysin and calmodulin. 1H n.m.r. spectra of the tetrapeptide are presented and assigned for D2O and DMSO solutions to determine the conformational mobility. The resonance of the two aspartyl side chains could be completely analysed and the vicinal coupling (C alpha H-C beta H and NH-C alpha H) indicated that the free peptide has considerable conformational mobility. The Ca(II) complex generates a different 1H n.m.r. spectrum for the aspartyl resonances at neutral pH. The solution conformation of Pr(III) complex of Ac-DVDA has been investigated using induced chemical shifts. The observed trends in the magnitude of the shift ratios and the rotamer population suggest that the metal ion binds predominantly to both carboxylates of two aspartyl residues in a bidentate fashion. We discuss the consistency of the differentiated spectra for aspartyl residues in the complex with the stepwise binding of Ca2+ to the carrier.     

Incorporation of labelled glycine into reduced glutathione of intact human erythrocytes by enzyme-catalysed exchange. A nuclear-magnetic-resonance study.

1H, 2H and 15N n.m.r. spectroscopy was used to monitor the incorporation of free glycine into the glycine residue of reduced glutathione (GSH) in suspensions of intact human erythrocytes. The following results were obtained. (i) By using 1H spin-echo n.m.r. the exchange reaction between [2H5]glycine and the protonated glycine residue of GSH was studied at various [2H5]glycine concentrations, thus enabling the calculation of an apparent Michaelis constant (Km) and maximal velocity (Vmax.) for the process. (ii) The reaction is catalysed by glutathione synthetase and proceeds most rapidly in the absence of glucose, which is the main physiological energy source of the erythrocyte. (iii) 15N n.m.r. spectroscopy, with a one-pulse sequence, and 2H n.m.r. spectroscopy, with an inversion recovery method, enabled demonstration of the incorporation of labelled glycine into an intra-erythrocyte peptide, consistent with incorporation into GSH. (iv) The exchange reaction, although inhibited by glucose, appeared not to be dependent on low ATP or 2,3-bisphosphoglycerate concentrations.     

N.m.r. studies of myelin basic protein. Conformation of a peptide that is an antigenic determinant for B-cell reactivity.

The peptide Gly-Arg-Ala-Ser-Asp-Tyr-Lys-Ser, derived from myelin basic protein (MBP), is part of an epitope to monoclonal antibodies to human MBP. Its conformation has been studied in aqueous solution by high-resolution one- and two-dimensional 1H and 13C n.m.r. Two-dimensional correlated spectroscopy, pH titrations and one-dimensional spin-decoupling techniques were employed to assign the spectra observed from both nuclei. Amide proton temperature coefficients, coupling constants, 13C spin-lattice relaxation times and nuclear-Overhauser-effect data provide evidence that the solution conformations of the octapeptide include a type-II beta-turn with a hydrogen bond between the CO group of Arg2 and the NH group of Asp5. The results are discussed in view of a possible conformation of the antibody receptor site.     

DL-apiose substituted with stable isotopes: synthesis, n.m.r.-spectral analysis, and furanose anomerization

The branched-chain pentose DL-apiose has been synthesized in good yield by a new and simple chemical method that can be adapted to prepare (1-13C)-, (2-13C)-, (1-2H)- and/or (2-2H)-enriched derivatives. N.m.r. spectra (1H- and 13C-) have been interpreted with the aid of selective (13C)- and (2H)-enrichment, and 2D and 13C[13C]-n.m.r. spectra. The solution composition of DL-(1-13C)apiose in 2H2O, determined by 13C-n.m.r. spectroscopy, has been found to differ from that determined previously by 1H-n.m.r. spectroscopy. Several 13C-1H and 13C-13C couplings have been measured and interpreted in terms of apiofuranose ring conformation. Ring-opening rate-constants of the four apiofuranoses [3-C-(hydroxymethyl)-alpha- and -beta-D-erythrofuranose, and 3-C-(hydroxymethyl)-alpha- and -beta-L-threofuranose] have been determined by 13C-saturation-transfer n.m.r. spectroscopy, and compared to those obtained previously for the structurally related tetrofuranoses.     

Conformations of dehydrophenylalanine containing peptides. Nuclear Overhauser effect study of two acyclic tetrapeptides

Two isomeric, acyclic tetrapeptides containing a Z-dehydrophenylalanine residue (delta Z-Phe) at position 2 or 3, Boc-Leu-Ala-delta Z-Phe-Leu-OMe (1) and Boc-Leu-delta Z-Phe-Ala-Leu-OMe (2), have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r. spectroscopy. In peptide 1 the Leu(4) NH group appears to be partially shielded from solvent, while in peptide 2 both Ala(3) and Leu(4) NH groups show limited solvent accessibility. Extensive difference nuclear Overhauser effect (n.O.e.) studies establish the occurrence of several diagnostic inter-residue n.O.e.s (Ci alpha H----Ni+1H and NiH----Ni+1H) between backbone protons. The simultaneous observation of "mutually exclusive" n.O.e.s suggests the presence of multiple solution conformations for both peptides. In peptide 1 the n.O.e. data are consistent with a dynamic equilibrium between an -Ala-delta Z-Phe- Type II beta-turn structure and a second species with delta Z-Phe adopting a partially extended conformation with psi values of +/- 100 degrees to +/- 150 degrees. In peptide 2 the results are compatible with an equilibrium between a highly folded consecutive beta-turn structure for the -Leu-delta Z-Phe-Ala- segment and an almost completely extended conformation.     

1H- and 13C-n.m.r. studies of the antitumour antibiotic luzopeptin. Resonance assignments, conformation and flexibility in solution.

The depsipeptide DNA-intercalating antibiotic luzopeptin was studied in solution by n.m.r. methods. Two-dimensional 1H double-quantum-filtered correlation spectroscopy (DQF-COSY) and nuclear-Overhauser-effect spectroscopy (NOESY) confirm the primary structure and twofold symmetry of luzopeptin and provide details of its three-dimensional conformation in solution. Trans-annular hydrogen bonds between the glycine NH groups and carbonyl oxygen atoms have been identified in the crystalline state [Arnold & Clardy (1981) J. Am. Chem. Soc. 103, 1243-1244], and are important in maintaining an antiparallel beta-sheet conformation. The n.m.r. data indicate that the glycine NH protons are appreciably shielded from the solvent molecules, which suggests that these hydrogen bonds are maintained in solution. The orientation of the quinoline chromophores is defined by two-dimensional NOE cross-peaks that position the N-methyl group of the L-beta-hydroxyvaline residue close in space to both the quinoline H-8 and serine NH proton. This pattern of NOEs is in accord both with the chromophore configuration found in the crystal and one where the quinoline rings are aligned in a parallel manner at right-angles to the depsipeptide ring. The n.m.r. data are consistent with a hydrogen bond between the quinoline hydroxy groups and the quinoline carbonyl oxygen atoms. The pyridazine acetylmethyl groups give NOEs to the C(alpha)H groups of the beta-hydroxy-N-methylvaline residues, showing that the acetyl groups, for at least some of the time, stretch over the depsipeptide ring, occluding one face of the molecule. Both of the latter features are also found in the crystal structure. Resonances in the 13C-n.m.r. spectrum of luzopeptin have been assigned by transferring 1H assignments to their covalently bonded carbon atoms via a heteronuclear shift-correlation experiment (HETCOR). The measurement of spin-lattice relaxation times and 1H-13C NOEs at specific sites in the molecule has led us to conclude that segmental motions within the depsipeptide ring are restricted and that the 13C relaxation data for luzopeptin's protonated carbon atoms are adequately described by isotropic tumbling in solution. Furthermore, relaxation data for the carbon atoms of the quinoline chromophores show that these rings exhibit similar motion to the depsipeptide ring and are not rotating rapidly with respect to it. Taken together all the data imply that luzopeptin is fairly rigid in solution, on the time scale of molecular tumbling, and has, or can readily attain, a staple-like structure suitable for bisintercalation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure and solution conformations of a cyclic trisaccharide from high-resolution n.m.r. spectroscopy and molecular modelling.

The conformational properties of a cyclic trisaccharide: [O-beta-D-glucopyranosyl-(1----6)]3 1,6"-anhydride nonacetate (C36H48O24, 1) have been established by high-resolution 1H- and 13C-n.m.r. spectroscopy in conjunction with potential-energy and molecular-mechanics calculations. The n.m.r. parameters used were nuclear Overhauser enhancements (n.O.e.) and coupling constants. From theoretical models of the trisaccharide, a statistical-mechanics approach was used to compute an ensemble average-relaxation matrix from which the n.O.e. were calculated. The observed nuclear Overhauser enhancements as measured by n.m.r. spectroscopy may be satisfactorily modelled if averaging over two conformational states is considered. In solution, both conformations of the molecule exhibit three-fold symmetry; the beta-linked glucopyranose rings have the 4C1 conformation. In one conformer, the orientation about the (1----6) linkage is characterized by torsion angles phi = 79.5 degrees, psi = 143.5, and omega = -64.3. For the other conformer, these values are phi = -137.7, psi = 68.2, and omega = 45.6. The existence of such a conformer shows that solution behaviour is not dominated by the stabilizing influence of the exoanomeric effect.     

Study of the phosphorylatable light chains of skeletal and gizzard myosins by nuclear magnetic resonance spectroscopy.

31P and 1H n.m.r. studies of the phosphorylatable light chains from rabbit fast skeletal and chicken gizzard muscles in the isolated state and in the intact myosin molecule indicate that the N-terminal region of the light chain containing the sites of phosphorylation has independent segmental flexibility. The ionization behaviour of serine phosphate in both rabbit skeletal and chicken gizzard P light chains exhibits cooperativity and is compatible with the phosphate group being influenced by neighbouring positively charged side-chains. No marked difference in phosphate ionization behaviour was apparent between the monophosphorylated P light chains of rabbit skeletal and chicken gizzard myosins. From 1H and 31P n.m.r. studies of the overall conformation, side-chain ionization properties and the spectral effects of titration with an anionic paramagnetic reagent bound at the basic N-terminal region, it is concluded that Thr-18 and Ser-19 are phosphorylated in the bisphosphorylated P light chain of gizzard myosin, the latter residue being the site of monophosphorylation. In the presence of F-actin the mobility of the serine phosphate of the P light chain of intact gizzard myosin was reduced. No interaction between the isolated P light chain and F-actin was however detected. These results are discussed with reference to the observed conformational features of the P light chain.     

The conformation of abscisic acid by n.m.r. and a revision of the proposed mechanism for cyclization during its biosynthesis.

The n.m.r. spectrum of abscisic acid (ABA) formed from [1,2-13C2]acetate by the fungus Cercospora rosicola shows 13C-13C coupling between C-6' (41.7 p.p.m.; 36 Hz) and the downfield 6'-methyl group (6'-Me) (24.3 p.p.m, 36 Hz). This 6'-Me, therefore, is derived from C-3' of mevalonate [Bennett, Norman & Maier (1981) Phytochemistry 20, 2343-2344]. An i.n.e.p.t. (insensitive nuclei enhanced by polarization transfer) pulse sequence demonstrated that the downfield 13C signal is produced by the 6'-Me that gives rise to the upfield 1H 6'-Me signal (23.1 d). The absolute configuration of this, the equatorial 6'-Me group, was determined as 6'-pro-R by decoupling and n.O.e. (nuclear-Overhauser-enhancement) experiments at 300 MHz using ABA, ABA in which the axial 6'-pro-S 5'-hydrogen atom had been exchanged with 2H in NaO2H and the 1',4'-cis- and 1',4'-trans-diols formed from these samples. The configuration at C-1' and at C-6' are now compatible with a chair-folded intermediate during cyclization, as proposed for beta- and epsilon-rings of carotenoids. ABA in solution exists, as in the crystalline form, with the ring in a pseudo-chair conformation. The side chain is axial and the C-3 Me and the C-5 hydrogen atoms are predominantly cis(Z).     

Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy

A series of nine closely related somatostatin analogues, containing the hexapeptide H-Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7-NH2 sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201-995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D-n.m.r. methods, in particular NOESY experiments and detection of long-range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' beta turn involving amino acids Phe3 to Thr6. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe1 and Thr8 (ol) plays a major role in stabilization of the active conformation.     

Assignments of the 1H- and 13C-n.m.r. spectra of tobramycin at low and high pH

The 1H-n.m.r. spectra (200 MHz) of protonated (pD 3.9) and non-protonated (pD 11.9) tobramycin have been analysed completely by 2D methods. The 3JH,H values are consistent with an essentially undistorted 4C1 conformation for each of the three moieties and are practically independent of the state of protonation. The resonances in the 13C-n.m.r. spectrum (50 MHz) have been reassigned at both pD values on the basis of 2D1H-13C chemical-shift correlation and 1D selective INEPT measurements.     

Statine and its derivatives. Conformational studies using nuclear magnetic resonance spectrometry and energy calculations

The conformations of derivatives of 3(S)-hydroxy-4(s)-amino-6-methylheptanoic acid (statine) and its analogs have been studied by n.m.r. in chloroform and in dimethyl sulfoxide, and by molecular mechanics calculations. The data obtained from these studies indicate that: 1) the coupling constant between NH and C4H is large, suggesting that the dihedral angle (theta) is near 165 degrees or 0 degree; 2) the coupling constant between C4H-C3H is small, indicating a vicinal bond angle of approximately 90 degrees; 3) the hydrogen deuterium exchange rate of statine amide protons is slow; however, the rate is dependent upon the electron withdrawing substituents adjacent to the amide NH's; 4) intramolecular hydrogen bonds involving the NH of the statine amide group do not stabilize conformations of single amino acid derivatives. Based on the n.m.r. results, four possible conformations of Boc-statine-OMe in solution are possible. MM1 calculations indicate one conformation is especially likely.     

The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study.

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5'-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3'-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a 'melting' temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3'-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5 X 10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.     

Crystal structure and n.m.r. analysis of lactulose trihydrate.

The 13C CPMAS n.m.r. spectrum of 4-O-beta-D-galactopyranosyl-D-fructose (lactulose) trihydrate, C12H22O11.3 H2O, identifies the isomer in the crystals as the beta-furanose. This is confirmed by a crystal structure analysis, using CuK alpha X-ray data at room temperature. The space group is P212121, with Z = 4 and cell dimensions a = 9.6251(3), b = 12.8096(3), c = 17.7563(4) A. The structure was refined to R = 0.031 and Rw 0.025 for 1929 observed structure amplitudes. All the hydrogen atoms were unambigously located on difference syntheses. The conformation of the pyranose ring is the normal 4C1 chair and that of the furanose ring is 4T3. The 1----4 linkage torsion angles are O-5'-C-1'-O-1'-C-4 = 79.9(2) degrees and C-1'-O-1'-C-4-C-5 = -170.3(2) degrees. All hydroxyls, ring and glycosidic oxygens, and water molecules are involved in the hydrogen bonding, which consists of infinite chains linked together by water molecules to form a three-dimensional network. There is a three-centered intramolecular, interresidue hydrogen bond from O-3-H to O-5' and O-6'. The n.m.r. spectrum of the amorphous, dehydrated trihydrate suggests the occurrence of a solid-state reaction forming the same isomeric mixture as was observed in crystalline anhydrous lactulose, although the mutarotation of the trihydrate when dissolved in Me2SO is very slow.     

Sequence-specific 1H-n.m.r. assignments and peptide backbone conformation in rat epidermal growth factor.

The solution conformation of rat epidermal growth factor (EGF) has been investigated by proton n.m.r. techniques. Two-dimensional proton n.m.r. experiments have allowed sequential resonance assignments to be made for most protons. On the basis of these assignments, two regions of anti-parallel beta-sheet structure have been derived from the n.m.r. data. A beta-sheet segment running from about V19 to V23 (capital letters refer to amino acids in the single-letter notation) is folded onto a beta-sheet segment running from R28 to N32 and joined by a chain reversal from E24 to D27. A second region involves a beta-turn from V34 to Y37, which starts a short beta-sheet up to G39, followed by a chain reversal up to Q43, which leads to folding of the C-terminal beta-sheet segment, i.e. H44-R45, running antiparallel to the short Y37 beta-sheet segment. The N-terminal segment up to G18 exists in a multiple bend conformation and is folded on to the V29-V23/R28-N32 beta-sheet such that Y10, Y13, Y22 and Y29 are proximal to each other. Structural comparison of rat, murine and human EGFs indicates a number of highly conserved structural features common to at least these species of EGF.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing alpha-aminoisobutyric acid

The conformation in aqueous solution of several alpha-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and 1H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro2 and/or Pro3 in BK stabilizes a degree of beta-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB7]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a beta-turn involving their common Arg1-Pro2-Pro3-Gly4 moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis' Pro and the propensity for a beta-turn at the N-terminus of the peptide.     

Conformation of the eosinophil chemotactic tetrapeptides and analogues in dimethyl sulfoxide. 1H n.m.r. studies

Proton nuclear magnetic resonance parameters are reported for DMSO-d6 solutions of the eosinophil chemotactic tetrapeptides, Val1-Gly2-Ser3-Glu4 and Ala1-Gly2-Ser3-Glu4, as well as three analogues of the Val1 tetrapeptide, D-Val1, Ala2 and Ala3. The synthesis of Val-(S)-[alpha-2 H1] Gly-Ala-Glu, in which the glycine has been stereospecifically deuterated in the H alpha 3 position, has allowed the assignment of the 1H resonances belonging to individual H alpha 2 and H alpha 3 glycine methylene protons. Simulation of the glycine ABX spin system yields two vicinal coupling constants which are consistent with a highly preferred conformation about the glycine HN-C alpha bond. The chemical shifts, coupling constants, temperature coefficients of amide proton chemical shifts and calculated side chain rotamer populations are reported for all peptides. The coupling constant analysis and temperature coefficients of amide proton chemical shifts together suggest that a type I beta-turn conformation is preferred by the Ala3 analogue. The 1H n.m.r. parameters of the other peptides suggest that these can also adopt a beta-turn conformation in DMSO. There are, however, considerable differences in the extent of conformational averaging undergone by the various peptides.     

N.m.r. studies of red cells

Recent n.m.r. studies of intact red cells are described. With 1H n.m.r. the normal high resolution spectra of red cells, even at high fields, are relatively uninformative because the very large number of resonances from the cells merge into a broad envelope. If a simple 90-tau-180 degree spin echo pulse sequence is used, however, many resonances can all be resolved. These include signals from haemoglobin histidines, glutathione, lactate and pyruvate. 13C and 31P signals have also been seen with a spectrometer converted to observe these nuclei essentially simultaneously. N.m.r. is well suited to monitor the time course of events after a perturbation of the cell system. Lactate increase, glutathione recovery after oxidation and alkylation of glutathione by iodoacetate can all be observed directly in red cell suspensions by means of 1H spin echo n.m.r. This method has also been used to measure isotope exchange (1H-2H) of lactate and of pyruvate at both the C-3 and the C-2 positions, and some of these exchange rates can be interpreted in terms of the activity of specific enzymes in the cells. 1H spin echo n.m.r. has also been used to obtain information about the transport rates of small molecules into cells. By means of the 13C/31P spectrometer and [13C-1] glucose, the 13C enrichment of 2,3-diphosphoglycerate (2,3-DPG) can be monitored at the same time as the levels of 2,3-DPG, ATP and inorganic phosphate are observed by 31P n.m.r.     

The fine structure of two DNA dodecamers containing the cAMP responsive element sequence and its inverse. Nuclear magnetic resonance and molecular simulation studies.

1H and 31P n.m.r. (nuclear magnetic resonance) spectroscopy have been used in conjunction with molecular simulation to determine the structure of two DNA dodecamers. The first of these, CATGACGTCATG, contains the octameric sequence CRE (cAMP responsive element), while the second is the reversed sequence, GTACTGCAGTAC. Structure determination was based on both NOESY (nuclear Overhauser spectroscopy) derived distances and COSY (correlated spectroscopy) dihedral angle data. Access to the 31P spectra also allowed the epsilon backbone angles to be determined. Considerable care was taken in deriving structural parameters from the n.m.r. data and an excellent level of agreement is obtained with the simulated conformations. Both dodecamers are found to belong to the B-DNA family; however, there is a striking difference between the CRE sequence and its inverse, the former conformation alone showing a strong structural heterogeneity.