Relaxation matrix analysis of two-dimensional nuclear Overhauser effect spectra

The large number of interproton distances extracted from two-dimensional nuclear Overhauser effect spectra has enabled determination of biomolecular structures in solution. The accuracy of those distances is increased substantially and the number of distances increased significantly by analysis of the experimental peak intensities using a complete relaxation matrix approach. More distances and more accurate distances both lead to a higher resolution structure. A complete relaxation matrix analysis also enables simulation of peak intensities for any postulated structure; comparison of these intensities with experimental intensities can provide a guide for structure refinement as well as a measure of the quality of the structure derived.     

Conformational analysis of protein side chains using two-dimensional Overhauser nuclear effect spectroscopy]

The method has been proposed to determine the conformations of protein side-chains (dihedral angles chi 1) using two-dimensional nuclear Overhauser effect spectroscopy data. This method is grounded of the algorithm prepared on the basis of joint consideration of proton-proton distance dependences in dipeptide units of L-amino acid residues on the dihedral angles phi, psi and chi 1 with the accounting of the local sterical conditions of the polypeptide chain. The obtained results gave the possibility to bring the different regions of space (phi, psi) of amino acid residues into the line with the specific sets of nuclear Overhauser effect spectral parameters which unambiguously characterize in most cases the conformational states of their side-chains. The method efficiency was displayed on the test calculation with the utilization as the experimental data of the "model" nuclear Overhauser effect contacts derived from the X-ray atomic coordinates of the bovine pancreatic trypsin inhibitor molecule.     

Local structure of cytochrome c from horse heart in solution. Conformational analysis using data of two-dimensional nuclear Overhauser effect spectroscopy]

Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between the conformations of two molecule forms are shown to be localized within the polypeptide chain fragments situated in the spatial structure near the heme crevice. The comparison of the dihedral phi and psi angles in the calculated conformations of horse cytochrome C with the corresponding characteristics of X-ray structures of tuna ferri- and ferrocytochrome c made for the oxidized and reduced protein forms using the quantitative criteria testifies the similarity of their conformations in solution and crystal. In is shown that the conformational changes of the separate amino acid residues which take place as the result of the "solution-to-crystal" transition occur on the surface fragments of protein globule and do not lead to essential alterations of the secondary molecule structure.     

Conformation of oligonucleotides in solution from data of the nuclear Overhauser effect

In this review the results of studies of oligonucleotides conformation in solution by 1D and 2D NOE over the last four years are presented. Theoretical basis of 2D NOE and other 2D techniques are briefly considered. The few attempts of determination of oligonucleotides structures in solution on the half-quantitative level and the perspectives of using 2D NOE for quantitative structure resolution are discussed.     

Method of determining protein conformations by the two-dimensional nuclear Overhauser enhancement spectroscopy data

A method is suggested to determine the most probable values of the angles phi, psi of the protein backbone by the data on the availability and absence of d connectivities in the two-dimensional nuclear Overhauser enhancement spectra. In view of this, the dependences of the proton-proton distances in dipeptide units of L-amino acid residues on the dihedral angles phi, psi, chi1 are considered and the conformational states of amino acid residues of the proteins with the known spatial structure are analysed statistically. The potentialities of the method are assessed with the aid of model spectral nuclear magnetic resonance (NMR) parameters obtained from the X-ray data for the bovine pancreatic trypsin inhibitor and avian pancreatic polypeptide. It is shown that the developed procedure of structural interpretation of the NMR data allows one to correctly reproduce the local conformation of the protein backbone. The obtained backbone conformation may serve as a starting point to build and refine molecular three-dimensional structure.     

Effects of internal motions on the development of the two-dimensional transferred nuclear Overhauser effect

Summary In this paper we address the influence of internal motions on the development of the transferred nuclear Overhauser effect in a ligand undergoing chemical exchange between a free and a bound state. We examine the effects of varying the effective correlation time as well as the motional order parameter for methyl group and phenyl ring rotations in the free and bound ligand conformations. The effect of decreasing the motional order for a proton pair on a methyl group or phenyl ring is to decrease the effective correlation time of the internuclear vector, and thus to decrease the cross-relaxation rate between the proton pair. This functions to dampen the effects of spin diffusion, especially in the bound ligand- where cross-relaxation rates are much faster than in the free ligand. The effect of changing the effective correlation time for methyl group motions has little effect on the build-up behaviour of the transferred nuclear Overhauser effect for small values of fraction bound, but a larger effect on how fast it decays. This effect is greater for internal motions in the free peptide than it is for internal motions in the bound peptide.Dedicated to the memory of Professor V.F. Bystrov     

Determination of the conformation of Lewis blood group oligosaccharides by simulation of two-dimensional nuclear Overhauser data

Through control of both the nmr probe temperature and of the solvent viscosity, phase-sensitive two-dimensional 1H nuclear Overhauser data (NOESY) at 300 and 500 MHz are obtained with excellent signal-to-noise ratios for Lewis blood group penta- and hexasaccharides isolated from human milk. Relatively long mixing times are required to produce measurable NOE intensities in these oligosaccharides, which makes a full relaxation matrix analysis necessary. By measurements of selective T1 for a few isolated 1H resonances, it was possible to generate a simulation of the complete NOESY spectrum at arbitrary mixing time for comparison with the experimental data. From an exhaustive search of the conformational space, it was found that only a small range of glycosidic dihedral angles of the nonreducing terminal Lewis blood group determinant fragments of the milk oligosaccharides LNF-2 and LND-1 produce simulated spectra agreeing within experimental error to the data. Conformational energy calculations reveal that each of these conformations is also one of minimum energy. It is concluded that the Lewis(a) and Lewis(b) oligosaccharides adopt relatively compact rigid structures in solution, as shown by the observation of cross peaks between protons in nonadjacent residues. Like the blood group A and H oligosaccharides, there exists only a small dependence of the conformation for Lewis(a) and Lewis(b) oligosaccharides on solvent. The apparent lack of dependence of conformation of these oligosaccharides on DMSO in D2O suggests that modification of solvent viscosity with mixtures of DMSO:D2O may provide a useful general strategy of NOESY studies of oligosaccharides.     

Structure refinement of a cyclic peptide from two-dimensional NMR data and molecular modeling

The conformational and dynamic properties of a cyclic peptide designed to inhibit human renin have been examined by using NMR and molecular modeling. From a quantitative analysis of a series of two-dimensional NOE data sets, proton-proton distances were calculated. Several different methods were explored and compared to incorporate these distance constraints as well as those derived from vicinal spin-spin coupling constants into computer-generated three-dimensional structures. These methods included interactive manual manipulation of the structures to fit the NMR-determined distance constraints, distance geometry, constrained energy minimizations, and constrained molecular dynamics. The advantages and disadvantages of the methods are discussed. In addition, to gain insight into the conformations accessible to the cyclic peptide and the relative flexibility of the different parts of the molecule, molecular dynamics calculations were performed at three different temperatures. Average interproton distances and dihedral angles were obtained from the structures generated in the dynamics trajectories and compared to those obtained from the NMR experiments. Despite the four methylene groups and ether linkage contained in the cyclic portion of the peptide, our NMR results indicated a preferred conformation for the macrocyclic ring of the peptide and supported the presence of a cis Phe-Ala peptide bond. In contrast, both the molecular dynamics and NMR data indicated a considerable amount of flexibility for the remaining noncyclic portion of the molecule. These results are used to propose an explanation for the cyclic peptide's inability to inhibit human renin.     

Direct NOE refinement of biomolecular structures using 2D NMR data

Summary A set of computer programs called DINOSAUR has been developed, which allows the refinement of biomolecular structures directly from 2D NOE intensities. The NOE restraining potential implemented emphasises the weak intensities corresponding to larger distances which are more likely to determine the three-dimensional structure. An approximation based on a two-spin approximation is proposed for the gradient of the NOE intensities instead of the exact solution which is extremely time-consuming. The DINOSAUR routines have been implemented in various refinement programs (Distance bound Driven Dynamics, Molecular Dynamics and Energy Minimisation) and tested on an eight-residue model peptide.     

Multiangular method for analysing molecular geometry from nuclear Overhauser effect results

A multiangular method, as an extension of a triangular method, has been developed in order to analyse the local conformation of a molecule in an atomic resolution from nuclear Overhauser effect results. When there is a rigid part in the molecule, and the nuclear Overhauser effect signals are observed between several spins attributed to the rigid part of the molecule and the target spin to be analysed, the geometrical probability density of the target spin can be found by the multiangulation method, using distances between spin pairs. The spin density is illustrated by a set of isograms similar to electron density maps from X-ray crystallographic analyses. The molecular model building is performed based upon the isograms. An application to the conformation analysis of transferred nuclear magnetic resonance results of NAD⁺, which binds to lactose dehydrogenace from Thermus caldophilus GK24, is described.     

Selective reversible deuteriation of oligodeoxynucleotides: simplification of two-dimensional nuclear Overhauser effect NMR spectral assignment of a non-self-complementary dodecamer duplex

Oligodeoxynucleotides are reversibly deuteriated at the purine C8 and cytosine C5 positions with deuterioammonium bisulfite at pD 7.8. The exchange reaction is complete after 48 h at 65 degrees C. When an oligomer deuteriated under these conditions is analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy, the purine H8 and cytosine H5 proton signals are selectively removed from the spectrum. A non-self-complementary oligodeoxynucleotide that has been deuteriated in this manner may be annealed with its complement and the resulting heteroduplex analyzed by two-dimensional nuclear Overhauser enhancement (NOESY) spectroscopy. NOE cross-peaks arising from pyrimidine H6-deoxyribose H1' dipolar interactions in both strands are observed, but purine H8-deoxyribose H1' and purine H8-deoxyribose H2',H2" dipolar interactions are only observed for the nondeuteriated strand. The intense cytosine H5-H6 cross-peaks are also removed from the spectrum of the deuteriated strand, which further simplifies interpretation since these strong cross-peaks often interfere with less intense NOE cross-peaks arising from dipolar coupling between purine H8 or pyrimidine H6 and deoxyribose anomeric protons. The resulting spectral simplification allows unambiguous assignments to be made on NOEs that otherwise may be difficult to distinguish. The deuteration procedure is demonstrated with the sequence d(CGTTATAATGCG).d(CGCATTATAACG), which has previously been assigned by traditional NOESY methods [Wemmer, D. E., Chou, S.-H., Hare, D. R., & Reid, B. R. (1984) Biochemistry 23, 2262-2268]. Although the assignment of this dodecadeoxynucleotide may be completed without deuteriation, several NOEs must be assigned indirectly because of degeneracies in the chemical shift of the purine H8 protons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of antibody aromatic residues with a peptide of cholera toxin observed by two-dimensional transferred nuclear Overhauser effect difference spectroscopy

The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE). Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imino-proton resonances of yeast tRNAPhe studied by two-dimensional nuclear Overhauser enhancement spectroscopy

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H2O solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G1 X C72 and C2 X G71 should be reversed). The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.     

Localization of hydrophobic ions in phospholipid bilayers using 1H nuclear Overhauser effect spectroscopy

The binding location for the hydrophobic ions tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-) was studied in sonicated phosphatidylcholine (PC) vesicles by measuring time-dependent and steady-state intermolecular 1H nuclear Overhauser effects (NOE's). Intermolecular cross-relaxation was also investigated by two-dimensional NOE spectroscopy. Information on the distance and order parameter dependence of the NOE's was obtained from a simple simulation of the NOE's in the alkyl chain region. Taken together, the NOE data and the simulation provide strong evidence that TPB- and TPP+, at low concentrations (less than or equal to 10 mol%), are localized in the alkyl chain region of the bilayer. At these lower concentrations of TPP+ or TPB-, no significant effect on lipid 13C T1 or T2 relaxation rates is detected. The proposed location is consistent with the expected free energy profiles for hydrophobic ions and with the carbonyl oxygens or interfacial water as the source of the membrane dipole potential. At higher ion/lipid ratios (greater than or equal to 20 mol%), TPB-/lipid NOE's increase. This results from a specific association of TPB- with the choline head group.     

Principles of quantitative analysis of two-dimensional spectra of nuclear Overhauser effect in evaluation of protein and peptide conformation

To elucidate potentialities of two-dimensional homonuclear Overhauser effect (NOESY) spectra of peptides and proteins for their spatial structure determination, impact of experimental parameters and intrinsic properties of the investigated molecule on proton cross-peak volumes in NOESY spectra was analysed. Recommendations which could increase accuracy of cross-peak volume measurements were suggested. Influence of intrinsic properties of a molecule (spin-lattice relaxation times T1, correlation time tau C and surrounding protons) on the volume of cross-peak for particular protons was analyzed using a complete relaxation matrix of the (formula; see text) helix of gramicidin A. Nonselective relaxation time T1 of the protons was found to affect only slightly the results of cross-peak volumes computer simulation, whereas correlation time tau C and surrounding protons seriously influenced cross-peak volumes. Nevertheless, cross-peak volumes between NH, C alpha H and C beta H protons of a dipeptide fragment of the entire molecule could be accurately simulated using the relaxation matrix of the individual dipeptide. Thus local conformations (torsion angles phi, psi and chi 1) of amino acid residues could be deduced independently of one another and prior to the complete analysis of a molecular structure. The result can be obtained even in the presence of spin-diffusion at mixing times providing maximal volumes of cross-peaks in NOESY spectra.     

Identification of localized redox states in plant-type two-iron ferredoxins using the nuclear Overhauser effect

The homonuclear Overhauser effect (NOE), in conjunction with nonselective spin-lattice relaxation measurements, has been employed to assign the contact-shifted resonances for the reduced form of two typical plant-type two-iron ferredoxins from the algae Spirulina platensis and Porphyra umbilicalis. These results demonstrate that the NOE should have broad general applicability for the assignments and electronic structural elucidation of diverse subclasses of paramagnetic iron-sulfur cluster proteins. NOE connectivities were detected only among sets of resonance exhibiting characteristically different deviations from Curie behavior, providing strong support for the applicability of the spin Hamiltonian formulation for the NMR properties of the antiferromagnetically coupled iron clusters [Dunham, W. R., Palmer, G., Sands, R. H., & Bearden, A. J. (1971) Biochim. Biophys. Acta 253, 373-384; Banci, L., Bertini, I., & Luchinat, C. (1989) Struct. Bonding (in press)]. The geminal beta-methylene protons for the two cysteines bound to the iron(II) center were clearly identified, as well as the C alpha H and one C beta H for each of the cysteines bound to the iron(III). The identification of the iron bound to cysteines 41 and 46 as the iron(II) in the reduced protein was effected on the basis of dipolar contacts between the bound cysteines, as predicted by crystal coordinates of S. platensis Fd [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. (Tokyo) 90, 1763-1773].(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation of double-stranded polydeoxynucleotides in solution by proton two-dimensional nuclear Overhauser enhancement spectroscopy

Proton 2D-NOE spectroscopy has been used to investigate the three-dimensional conformations of several sonicated polydeoxynucleotides in solution. The observed pattern of cross peaks indicate that poly(dA-dT) · poly(dA-dT) in all salt concentrations studied (up to 6.6M CsF), and poly(dG-m⁵dC) · poly(dG-m⁵dC) in low salt (0.1M NaCl) are righthanded B-structures. Poly(dG-m⁵dC) · poly(dG-m⁵dC) in Mg²⁺ (3 mM) solution exhibits a pattern characteristic of the left-handed Z-form. These results for poly(dA-dT) · poly(dA-dT) are in contrast to suggestions that this copolymer exists as a left-handed form, either in low or high salt. We present pure absorption-mode 2D-NOE spectra that enable us to compare several distances and define the conformations of these polydeoxynucleotides in solution.     

Investigation of the solution structure of a DNA octamer [d(GGAATTCC)]2 using two-dimensional nuclear Overhauser enhancement spectroscopy

Proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra in the pure absorption phase were obtained at 500 MHz for [d(GGAATTCC)]2 in aqueous solution at a series of mixing times. The experimental data were analyzed by comparison with theoretical spectra calculated using the complete 70 X 70 relaxation matrix including all proton dipole-dipole interactions and spin diffusion [Keepers, J. W. & James, T. L. (1984) J. Magn. Reson. 57, 404-426]. The theoretical spectra at each mixing time were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on the similarity of the six internal residues of the title octamer with those of the dodecamer [d(CGCGAATTCGCG)]2, for which the crystal structure has been determined. Neither the standard B-form nor the energy-minimized structure will yield theoretical 2D NOE spectra which accurately reproduce all peak intensities in the experimental spectra. However, many features of the experimental spectra can be represented by both the B-form and the energy-minimized structure. Sequence-dependent structural characteristics are manifest in the 2D NOE spectra, in particular at the purine-pyrimidine junction as noted previously in the crystal structure. On the whole, the energy-minimized structure appears to yield theoretical 2D NOE spectra which mimic many, if not all, aspects of the experimental spectra. All 2D NOE data were consistent with nanosecond correction times as implied by proton spin-lattice relaxation time measurements. But better fits of some of the 2D NOE data using small variations in an effective isotropic correlation time suggest that there may be some local variations in mobility within the octamer duplex structure in solution.     

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].     

Determination of the chirality of the saturated pyrrole in sulfmyoglobin using the nuclear Overhauser effect

The interproton nuclear Overhauser effect (NOE) and paramagnetic dipolar relaxation rates for hyperfine-shifted resonances in the proton NMR spectra of sperm whale met-cyano sulfmyoglobin have led to the location and assignment of the proton signals of the heme pocket residue isoleucine 99 (FG5) in two sulfmyoglobin isomers. Dipolar relaxation rates of these protein signals indicate a highly conserved geometry of the heme pocket upon sulfmyoglobin formation, while the similar upfield direction of dipolar shifts for this residue to that observed in native sperm whale myoglobin reflects largely retained magnetic properties. Dipolar connectivity of this protein residue to the substituents of the reacted heme pyrrole ring B defines the stereochemistry of the puckered thiolene ring found in one isomer, with the 3-CH3 tilted out of the heme plane proximally. The chirality of the saturated carbons of pyrrole ring B in both the initial sulfmyoglobin product and the terminal alkaline product is consistent with a mechanism of formation in which an atom of sulfur is incorporated distally to form an episulfide across ring B, followed by reaction of the vinyl group to yield the thiolene ring that retains the C3 chirality.