Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy.

Conformations of dibucaine and tetracaine in small unilamellar phosphatidylcholine vesicles have been investigated by nuclear Overhauser effects (NOEs) in 1H nuclear magnetic resonance spectroscopy. Two-dimensional NOE and chemical exchange correlated spectroscopy (NOESY) and rotating frame NOE spectroscopy (ROESY) methods have been applied for obtaining the NOEs. In the NOESY spectra, NOEs between protons within the drug were overwhelmed by spin diffusion even at a short mixing time. This observation reduced the usefulness of the NOESY method on the one hand, however, on the other hand it facilitated remarkably in revealing signals due to the drug, hidden in the broad resonances of the membranes. In the ROESY spectra, the spin diffusion phenomena were less effective; accordingly the conformations of the drugs interacting with membranes were determined by the ROESY method. The observed NOE data showed that dibucaine takes more than two conformations and that both dibucaine and tetracaine are present as a dimer in the membranes. Molecular dynamics calculations supported these findings.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

NOE and two-dimensional correlated 1H-NMR spectroscopy of cytochrome c' from Chromatium vinosum.

1H two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatium vinosum have been obtained. The protein is of medium size (Mr 28,000), essentially high spin (S = 5/2) although some quantum mechanical spin admixing with S = 3 2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (alpha-CH2 propionate and beta-CH2 protons of the bound histidine) and between alpha-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems.     

A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens.

Transferred nuclear Overhauser effect (TRNOE) spectroscopy can be used to study intra- and intermolecular interactions of bound ligands complexed with large proteins. However, the 2D NOE (NOESY) spectra of large proteins are very poorly resolved and it is very difficult to discriminate the TRNOE cross peaks, especially those due to intermolecular interactions, from the numerous cross peaks due to intramolecular interactions in the protein. In previous studies we measured two-dimensional difference spectra that show exclusively TRNOE and exchange cross-peaks (Anglister, J., 1990. Quart. Rev. Biophys. 23:175-203). Here we show that a filtering method based on the difference between the T1rho values of the ligand and the protein protons can be used to directly obtain a two-dimensional transferred NOE spectrum in which the background cross-peaks due to intramolecular interactions in the protein are very effectively removed. The usefulness of this technique to study protein ligand interactions is demonstrated for two different antibodies complexed with a peptide of cholera toxin (CTP3). It is shown that the T1 rho-filtering alleviates t problems encountered in our previous measurements of TRNOE by the difference method. These problems were due to imperfections in the subtraction of two spectra measured for two different samples.     

Micelle formation of bile salts and zwitterionic derivative as studied by two-dimensional NMR spectroscopy

The self-association of sodium taurodeoxycholate (NaTDC) and a zwitterionic derivative of cholic acid (CHAPS) in deuterium oxide was investigated by one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts of several protons suggested that NaTDC and CHAPS form nonamers and heptamers, respectively, as well as dimer. The equilibrium constants of dimerization and the micellar aggregation numbers are close to the literature values. From the intensities of intermolecular cross-peaks in the nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectra of NaTDC and CHAPS micellar solutions, partial structures of their micelles were estimated. The CHAPS micelle consists mainly of the back-to-back association, similarly to taurocholate (NaTC). However, the NaTDC micelle consists of the back-to-face association, because the face of NaTDC is rather hydrophobic. Furthermore, the back of bile molecules forms a convex plane and the face forms a concave plane. The back-to-face structure of NaTDC will be stabilized by a close contact between these planes. The chemical shift changes of several protons of CHAPS and NaTC in the micellar state are close to each other, but are different from those of NaTDC. This finding is consistent with the difference in their micellar structures.     

Conformation of a bulge-containing oligomer from a hot-spot sequence by NMR and energy minimization

Two-dimensional nmr data on a bulge-containing oligodeoxyribonucleotide, 5'dGATGGGCAG.dCTGACCCATC, and a regular oligomer of similar sequence, 5'dGATGGCAG.dCTGCCATC, are presented. The nonexchangeable protons are assigned from sequential nuclear Overhauser effect spectroscopy (NOESY) connectivities. The two-dimensional NOE (NOESY) and correlated (COSY) spectra of the bulge-containing oligomer are compared to those of the perfect 8-mer. Experimental proton-proton distances are determined from NOESY spectra acquired with mixing times of 100, 150, and 200 ms, using comparable distances in the B-DNA region of the molecule as a calibration. With this approach, measured distances do not depend systematically on mixing time. Energy minimization techniques are used to calculate a three-dimensional structure for the bulge-containing oligomer in agreement with the nmr data. The helix is of the B family, with the extra adenine stacked into the helix, and the helix axis is bent by 20 degrees.     

Sequence-specific solution structure of d-GGTACGCGTACC

Complete resonance assignments of nonexchangeable base protons and sugar protons in d-GGTACGCGTACC at 500 MHz have been obtained by two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The characteristic phase-sensitive multiplet patterns of the ntrasugar cross peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). These coupling constants combined with the intranucleotide COSY cross peak intensities have been used to identify the sugar pucker of individual nucleotide units. In most cases, the deoxyribose rings adopt a conformation close to O4'-endo. Spin-diffusion has been monitored from the buildup of the normalized volumes of NOE cross peaks in NOESY spectra as a function of mixing time. A set of 55 intranucleotide and internucleotide interproton distances have been estimated from the low mixing time NOESY spectrum (tau m = 75 ms). The estimated intranucleotide proton-proton distances have been used to determine the individual glycosidic dihedral angles of the nucleotide units which lie in the anti domain. It is observed that the molecule adopts an overall conformation close to that of the B-form although there are differences in the intricate details.     

Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances

The use of proton-proton nuclear Overhauser enhancement (NOE) distance information for identification of polypeptide secondary structures in non-crystalline proteins was investigated by stereochemical studies of standard secondary structures and by statistical analyses of the secondary structures in the crystal conformations of a group of globular proteins. Both regular helix and beta-sheet secondary structures were found to contain a dense network of short 1H-1H distances. The results obtained imply that the combined information on all these distances obtained from visual inspection of the two-dimensional NOE (NOESY) spectra is sufficient for determination of the helical and beta-sheet secondary structures in small globular proteins. Furthermore, cis peptide bonds can be identified from unique, short sequential proton-proton distances. Limitations of this empirical approach are that the exact start or end of a helix may be difficult to define when the adjoining residues form a tight turn, and that unambiguous identification of tight turns can usually be obtained only in the hairpins of antiparallel beta-structures. The short distances between protons in pentapeptide segments of the different secondary structures have been tabulated to provide a generally applicable guide for the analysis of NOESY spectra of proteins.     

Complete assignment of the 500 MHz 1H-NMR spectra of bleomycin A2 in H2O and D2O solution by means of two-dimensional NMR spectroscopy

1H-NMR spectra of bleomycin A2 recorded at 500 MHz in D2O and H2O at 24 degrees C and 3 degrees C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlation spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 113 exchangeable and 59 non-exchangeable protons in the 1H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3 degrees C and 24 degrees C suggest that at low temperatures the central party of the bleomycin A2 molecule tends to adopt an extended conformation.     

Contributions from hydration of carboxylate groups to the spectrum of water-polypeptide proton-proton Overhauser effects in aqueous solution

Summary Nuclear Overhauser effects (NOE) were measured between water protons and protons of the glutamic acid side chain of the bicyclic decapeptide in aqueous solution. Positive NOEs were observed between the CH₂ group of Glu and the water resonance, with similar NOE intensities at pH 2.0 and pH 6.3 in both the laboratory frame and the rotating frame of reference. These results indicate that the residence times of the hydration water molecules near the side-chain methylene protons are shorter than 500 ps for both the charged form and the uncharged form of Glu, and hence comparable to the water residence times near uncharged amino acid side chains. Furthermore, this study shows that the acidic proton in protonated carboxylic acid groups is not likely to interfere with the observation of polypeptide-hydration water NOEs, which is in contrast to the hydroxyl protons of the side chains of serine, threonine and tyrosine.Abbreviations NOE nuclear Overhauser effect - NOESY NOE spectroscopy in the laboratory frame - ROESY NOE spectroscopy in the rotating frame - ID one-dimensional - 2D two-dimensional - HPLC high-pressure liquid chromatography     

Study of the spatial structure of the duplex d(pTGTTTGGC) d(pCCAAAC)A in aqueous solution by methods of uni- and two-dimensional (1)H-NMR spectroscopy and organic molecular mechanics

Structure of the complementary complex d(pTGTTTGGC) d(pCCAAAC)A in the aqueous solution has been investigated by one- and two-dimensional 1H NMR spectroscopy. The resonances of nonexchangeable protons of bases as well as methyl and deoxyribose 1', 2'a, 2'b, 3' and 4' protons have been assigned by means of two-dimensional J-correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY). Using one-dimensional NOE measurements, 62 interproton distances (intranucleotide: (H6/H8)i--(H1')t, (H6/H8)i--(H2'a)i, (H1')i--(H2'a)i, (H1')i--(H2'b)i; internucleotide: (H6/H8)i--(H1')i-1, (H6/H8)i--(H2'a)i-1, (H6/H8)i--(H2'b)i-1, (H5/CH3)i--(H6/H8)i-1, (H5/CH3)i--(H2'a/H2'b)i-1) have been determined for nearest-neighbour protons. Spin-coupling constant values for some sugar protons have been obtained from COSY spectra. The restrained molecular mechanics calculations have yielded the possible solution structures of duplex fitting the experimental set of interproton distances and coupling constants.     

Spectroscopic evidence for a preferential location of lidocaine inside phospholipid bilayers

We examined the effect of uncharged lidocaine on the structure and dynamics of egg phosphatidylcholine (EPC) membranes at pH 10.5 in order to assess the location of this local anesthetic in the bilayer. Changes in the organization of small unilamellar vesicles were monitored either by electron paramagnetic resonance (EPR)-in the spectra of doxyl derivatives of stearic acid methyl esters labeled at different positions in the acyl chain (5-, 7-, 12- and 16-MeSL)-or by fluorescence, with pyrene fatty-acid (4-, 6-, 10- and 16-Py) probes. The largest effects were observed with labels located at the upper positions of the fatty-acid acyl-chain. Dynamic information was obtained by 1H-NMR. Lidocaine protons presented shorter longitudinal relaxation times (T(1)) values due to their binding, and consequent immobilization to the membrane. In the presence of lidocaine the mobility of all glycerol protons of EPC decreased, while the choline protons revealed a higher degree of mobility, indicating a reduced participation in lipid-lipid interactions. Two-dimensional Nuclear Overhauser Effect experiments detected contacts between aromatic lidocaine protons and the phospholipid-choline methyl group. Fourier-transform infrared spectroscopy spectra revealed that lidocaine changes the access of water to the glycerol region of the bilayer. A "transient site" model for lidocaine preferential location in EPC bilayers is proposed. The model is based on the consideration that insertion of the bulky aromatic ring of the anesthetic into the glycerol backbone region causes a decrease in the mobility of that EPC region (T(1) data) and an increased mobility of the acyl chains (EPR and fluorescence data).     

Consequences of methylation on the amino group of adenine. A proton two-dimensional NMR study of d(GGATATCC) and d(GGm6ATATCC)

A two-dimensional 500-MHz 1H-NMR study of two oligonucleotides, d(GGATATCC) and d(GGm6ATATCC), is presented in which we have investigated the effects of adenine methylation. The two-dimensional nuclear Overhauser spectra (NOESY) show that both oligonucleotides adopt a normal right-handed B-type helix and one-dimensional nuclear Overhauser enhancement (NOE) studies demonstrate that any difference in conformation must be small. However methylation drastically slows down the helix in equilibrium coil exchange which becomes slow on a proton NMR time scale. While d(GGATATCC) fits a two-site exchange model, d(GGm6ATATCC) does not and we invoke the presence of a third species which may be an intermediate in helix formation. NMR and ultraviolet spectroscopy show that methylation destabilizes the helix, measured by the melting temperature and enthalpy of dissociation.     

Quantification of DNA structure from NMR data: conformation of d-ACATCGATGT

Resonance assignments of nonexchangeable base and sugar protons have been obtained in double-helical d-ACATCGATGT by using two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The exchangeable imino protons have been assigned on the basis of their chemical shifts. The characteristic phase-sensitive multiplet patterns of the intrasugar cross-peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). The information on the J values combined with the intranucleotide COSY cross-peak intensities has been used to identify sugar puckers of individual nucleotide units. In most cases, the deoxyribofuranose rings are found to adopt a conformation close to O4'-endo. Spin diffusion has been monitored from the buildup of the normalized volumes of NOE cross-peaks in NOESY spectra as a function of mixing time. A set of 52 intranucleotide and internucleotide proton-proton distances have been estimated by using low mixing time NOESY spectra (tau m = 40 and 80 ms). The estimated intrasugar proton-proton distances rule out possibilities of existence of a fast equilibrium between C2'-endo and C3'-endo conformations. Intranucleotide proton-proton distances combined with the knowledge of sugar puckers have been used to fix the glycosidic bond torsion angle (chi). For this purpose, simulated distance contours depicting the dependence of intranucleotide proton-proton distances on pseudorotational phase angle (P) and glycosidic bond torsion angle (chi) have been used. Further, the proton homonuclear (J, delta) spectrum has been used to monitor the 31P-1H heteronuclear couplings, which are preserved in the omega 2 projection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two-dimensional 1H NMR of gene 5 protein indicates that only two aromatic rings interact significantly with oligodeoxynucleotide bases

The interaction of gene 5 protein (G5P) with oligodeoxynucleotides is investigated by 1H NMR methods, principally two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Aromatic resonances of G5P are specifically assigned from crystallographic data, while the low-field resonances of nucleotides are assigned with sequential or other procedures. Chemical shift changes that accompany binding of d(pA)4, d(A)4, d(pT)4, and d(pA)8, combined with specific protein-nucleotide nuclear Overhauser effects (NOEs) obtained from NOESY spectra, suggest that Phe-73 and Tyr-26 are the only aromatic residues that stack significantly with nucleotide bases. Chemical shift data also imply a role for Leu-28, though this has not been confirmed with intermolecular NOEs. Binding of all four oligonucleotides causes marked upfield movements (0.1-0.6 ppm) of G5P NOESY cross peaks belonging to Tyr-26, Leu-28, and Phe-73. Most other G5P spin systems, notably those of Tyr-34 and Tyr-41, do not appear to be significantly affected. In the d(pA)4-G5P complex an intermolecular NOE is observed between Tyr-26 and H1' of Ade-1, while Phe-73 has NOEs with the H2, H8, and H1' protons of Ade-2 and -3. Intramolecular NOEs seem to follow a similar pattern in the partially cooperative d(pA)8-G5P complex, though specific nucleotide resonance assignments are not possible in this case. Binding causes relatively small chemical shift changes for the base resonances in adenylyl nucleotides, suggesting that there is some, but not complete, unstacking of the bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

反向DPPC脂囊泡中短杆菌肽S的二维氢核磁共振研究

本文用300MHz核磁共振仪对反向DPPC脂囊泡中短杆菌肽S进行了研究.用二维相关谱(COSY)和二维NOE谱(NOESY)对反向脂囊泡中短杆菌肽S的共振峰进行了识别,短杆菌肽S所有的共振峰都被指定.并在此基础上,通过NOESY及一维自旋回波谱对短杆菌肽S在反向脂囊泡中的构象进行了分析.结果表明,短杆菌肽S在反向脂囊泡中不再为平面环状结构,而是有某种程度的折叠.     

A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules

The recently developed technique of two-dimensional (2D) cross-relaxation spectroscopy is utilized for systematic measurements of selective nuclear Overhauser enhancements (NOE) in the high resolution ¹H nuclear magnetic resonance (NMR) spectra of biological macromolecules in solution. Compared to conventional one-dimensional NOE studies, the 2D NOE experiment has the principal advantage that it avoids detrimental effects arising from the limited selectivity of preirradiation in crowded spectral regions. Furthermore, it yields with a single instrument setting a complete network of NOE's between all the protons in the macromolecule. The resulting information on intramolecular proton-proton distances provides a new avenue for studies of the spatial structures of biopolymers.     

Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix

The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolve many ambiguities in intraresidue J-coupled connectivities and interresidue NOE connectivities. This required no explicit knowledge of the solution structure. The pattern of NOEs found is consistent with a regular alpha helix between glycine-6 and lysine-13; H bonding continues at least through alanine-15 [see Wand, A.J., Roder, H., & Englander, S. W. (1986) Biochemistry (following paper in this issue)]. Chain disorder occurs at the N-terminus. There is no indication of significant spin diffusion among the backbone amide and alpha-protons of this 12.4-kilodalton protein even at the longest NOE mixing time used (140 ms).     

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Locations and dynamical perturbations for lipids of cationic forms of procaine, tetracaine, and dibucaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Locations and dynamical perturbations for lipids of local anesthetics (procaine . HCl, tetracaine . HCl, and dibucaine . HCl) in sonicated egg yolk phosphatidylcholine (PC) vesicles have been studied by 1H-1H nuclear Overhauser effect (NOE) measurements. It was found that tetracaine and dibucaine bind much strongly to the neutral lipids than does procaine and that their mobilities are lowered to such an extent that spin diffusion is transmitted (i.e., omega 2 tau c2 much greater than 1). The intermolecular NOEs between drugs and PC were more effective in the case of dibucaine than with tetracaine, indicating that dibucaine binds to the lipids more strongly than tetracaine; this order agrees well with that of anesthetic potency. However, it was only tetracaine that gave any appreciable dynamical perturbation to the PC vesicles when they were monitored by the extent of transfer of the negative NOE from alpha-methylene protons to choline methyls, olefinic methines, acyl methylenes and terminal methyl protons. This finding was interpreted as being due to the differences in the locations of these drugs in small unilamellar vesicles: (1) procaine interacts with lipids very weakly at the outer surface of the vesicles; (2) tetracaine binds to the lipids both at the outer and inner halves of the bilayer, inserting its rod-like molecule in a forest of acyl chains of PC; (3) dibucaine binds tightly to the polar head-group of PC, which resides only at the outer half of the bilayer vesicles. It was concluded that the relative order of anesthetic potency within these drugs can be correlated not with the ability to affect membrane fluidity but with the ability to bind to lipids at the polar head-group of the bilayer vesicles.