Determination of local conformation of proteins from 1H-NMR spectroscopy data

A method is proposed to determine conformations of amino acid residues of the protein and effective correlation time tau c from cross-peak intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra. The method consists in fitting complete relaxation matrix of dipeptide unit protons to experimental cross-peak intensities by varying phi, psi, chi torsional angles and tau c. To verify the method, NOESY spectra of basic pancreatic trypsin inhibitor (BPTI) were theoretically generated at mixing times tau m = 25-300 ms and tau c = 4 ns and used for local structure determination. The method works well with optimum for measurement of NOE intensities tau m 100-200 ms. As a result, the backbone phi, psi torsion angles were unambiguously determined at tau m = 100 ms for all but Gly residues of BPTI, and chi 1 angles were determined for the majority of side chains. The obtained dipeptide unit conformations are very close to the BPTI crystallographic structure: root mean square deviation (RMSD) of interproton distances within dipeptide units, on the average, is 0.08 A (maximal deviation 0.44 A), and RMSD of phi and psi angles are 18 and 9 degrees, respectively (maximal deviations are 44 and 22 degrees).     

Determination of the local structure of the protein insectotoxin I5A from the scorpion Buthus eupeus from 1H-NMR spectroscopy data

The local structure (torsion angles phi, psi and chi 1 of amino acid residues) of insectotoxin I5A (35 residues) of scorpion Buthus eupeus has been determined from cross-peak integral intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra and spin coupling constants of vicinal H--NC alpha--H and H--C alpha C beta--H protons. The local structure determination was carried out by fitting complete relaxation matrix of peptide unit protons (protons of a given residue and NH proton of the next residue in the amino acid sequence) with experimental NOESY cross-peak intensities. The obtained intervals of backbone torsional angles phi and psi consistent with NMR data were determined for all but Gly residues. The predominant C alpha--C beta rotamer of the side chain has been unambiguously determined for 42% of the insectotoxin amino acid residues whereas for another 46% residues experimental data are fitted equally well with two rotamers. Stereospecific assignments were obtained for 38% of beta-methylene groups. The determined torsional angles phi, psi and chi 1 correspond to the sterically allowed conformations of the amino acid residues and agree with the insectotoxin secondary structure established earlier by 1H NMR spectroscopy.     

Refinement of the spatial structure of the gramicidin A ion channel]

The spatial structure of the gramicidin A (GA) transmembrane ion-channel was refined on the base of cross-peak volumes measured in NOESY spectra (mixing time tau m = 100 and 200 ms). The refinement methods included the comparison of experimental cross-peak volumes with those calculated for low-energy GA conformations, dynamic averaging of the low-energy conformation set and restrained energy minimization. Accuracy of the spatial structure determination was estimated by the penalty function Fr defined as a root mean square deviation of interproton distances corresponding to the calculated and experimental cross-peak volumes. As the initial conformation we used the right-handed pi 6,3 LD pi 6,3 LD helix established on the base of NMR data regardless of the cross-peak volumes. The conformation is in a good agreement with NOE cross-peak volumes (Fr 0.2 to 0.5 A depending on NOESY spectrum). For a number of NOEs formed by the side chain protons, distances errors were found as much as 0.5-2.0 A. Restrained energy minimization procedure had little further success. However some of these errors were eliminated by the change in torsional angle chi 2 of D-Leu12 and dynamic averaging of the Val7 side chain conformations. Apparently, majority of deviations of the calculated and experimental cross-peak volumes are due to the intramolecular mobility of GA and cannot be eliminated within the framework of rigid globule model. In summary the spatial structure of GA ion-channel can be thought as a set of low-energy conformations, differing by the side chain torsion angles chi 1 Val7 and chi 2 D-Leu4 and D-Leu10 and the orientation of the C-terminal ethanolamine group. Root mean square differences between the atomic coordinates of conformations are in the range of 0.3-0.8 A.     

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)     

NMR structural refinement of an extrahelical adenosine tridecamer d(CGCAGAATTCGCG)2 via a hybrid relaxation matrix procedure

Until very recently interproton distances from NOESY experiments have been derived solely from the two-spin approximation method. Unfortunately, even at short mixing times, there is a significant error in many of these distances. A complete relaxation matrix approach employing a matrix eigenvalue/eigenvector solution to the Bloch equations avoids the approximation of the two-spin method. We have calculated the structure of an extrahelical adenosine tridecamer oligodeoxyribonucleotide duplex, d(CGCAGAATTCGCG)2, by an iterative refinement approach using a hybrid relaxation matrix method combined with restrained molecular dynamics calculations. Distances from the 2D NOESY spectra have been calculated from the relaxation rate matrix which has been evaluated from a hybrid NOESY volume matrix comprising elements from the experiment and those calculated from an initial structure. The hybrid matrix derived distances have then been used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure is then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. Although the crystal structure of the tridecamer clearly shows the extrahelical adenosine looped out way from the duplex, the NOESY distance restrained hybrid matrix/molecular dynamics structural refinement establishes that the extrahelical adenosine stacks into the duplex.     

Spatial structure of (1-36)bacterioopsin solubilized in a methanol-chloroform mixture with sodium dodecylsulfate micelles]

Spatial structures of proteolytic segment A (sA) of bacterioopsin of Halobacterium halobium (residues 1-36) solubilized in the mixture of methanol-chloroform (1:1), 0.1 M LiClO4 or in perdeuteriated sodium dodecyl sulfate (SDS) micelles, were determined by 2D 1H-NMR techniques. Most of the resonances in 1H-NMR spectra of fragment A were assigned using DQF-COSY, TOCSY and NOESY spectra. Deuterium exchange rates for amide protons were measured in series of NOESY spectra. 324 and 400 NOESY cross-peak volumes were measured in NOESY spectra of sA in mixture of organic solvents and SDS micelles, respectively. The sA structure was determined by local structure analysis, distance geometry calculation with program DIANA and systematic search for energetically allowed side chain rotamers consistent with NOESY cross-peak volumes. The structures of sA are similar in both milieus. These structures have the right-handed alpha-helical region from Pro-8 to Met-32 with root mean square deviation (RMSD) of 0.25 A between back bone heavy atoms and fit well with Pro-8 to Met-32 alpha-helical region in electron cryo-microscopy (ECM) model of bacteriorhodopsin [4]. The C-terminal region Gly-33-Asp-36 is disordered in both milieus, while N-terminal region Ala-2-Gly-6 in organic solvents has a fixed structure (RMSD of 0.25 A) stabilized by the Thr-5 NH...O=C Gln-3 and Ile-4 NH...O = C Ala-2 hydrogen bonds. This region of sA in SDS micelles has disordered structure with RMSD of 1.44 A for back bone heavy atoms. Torsion angles chi 1 of sA were unequivocally determined for 72% of side chains in the alpha-helical region and are identical in both milieus.     

Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of a covalent CPI-CDPI2-oligodeoxyribonucleotide decamer complex

CPI-CDPI2 is a synthetic analogue of CC-1065, which is a naturally occurring antitumor antibiotic. Assignment of the 1H NMR spectra of a CPI-CDPI2-oligodeoxyribonucleotide decamer, d-(CGCTTAAGCG)2, complex has been made by two-dimensional 1H/1H spectroscopy. The solution structure of the complex was calculated by an iterative hybrid relaxation matrix method combined with NOESY distance restrained molecular dynamics. Refinement proceeded in two steps in which the decamer was initially refined alone and then CPI-CDPI2 was added to the structure to allow initial estimates of drug-DNA contacts. A hybrid matrix/MD refinement was used to better take into account problems associated with spin diffusion. Thus the distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experimental spectrum and those calculated from an initial structure. The hybrid matrix derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. The efficacy of CC-1065 has been attributed to its minor groove binding and alkylation to the N3 position of adenosine. CPI-CDPI2 appears to bind to the decamer in a similar manner. The effect of CPI-CDPI2 on the decamer's 1H and 31P spectrum was consistent with a minor groove binding motif with the drug alkylating at A17 with the CDPI rings oriented toward the 5'-end of the alkylated strand. In addition, the NMR data support one major adduct but also indicate the presence of a minor adduct. The latter could represent a drug alkylation of the DNA at a secondary site (or alternative orientation of the rings).     

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.     

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.     

NMR investigations of duplex stability of phosphorothioate and phosphorodithioate DNA analogues modified in both strands.

Duplex formation from the self-complementary 12mer d(CGCGAATTCGCG) (Dickerson dodecamer) in which all phosphodiester linkages were replaced by phosphorothioate or phosphorodithioate linkages was studied using variable-temperature 1H and 31P NMR spectroscopy. Melting temperatures of the dodecamer, measured spectrophotometrically, showed significant decrease upon sulfur substitution (Tm 49 degrees C for the phosphorothioate and 21 degrees C for the phosphorodithioate, compared with 68 degrees C for the unmodified oligomer, in 1 M salt). Hyperchromicity observed upon melting of the dithioate was surprisingly low. NOESY spectra of the monothioate showed a cross-peak pattern characteristic for a right-handed duplex. Imino proton resonances of the duplex, shown by the mono- and the dithioate, were similar to those of the parent compound. In spite of monophasic melting curves, temperature dependence of the imino proton resonances and phosphorus resonances of the phosphorodithioate indicated heterogeneity with respect to base-pairing, compatible with the presence of a hairpin loop. Relaxation times (T1) of the imino protons in the phosphorothioate, determined by the saturation recovery method, were considerably shorter than in the unmodified oligomer. Base-pair lifetimes in the unmodified Dickerson dodecamer, determined by catalyst-dependent changes in relaxation rates of imino protons, were in the range of 2-30 ms at 20 degrees C. Strongly reduced base-pair lifetimes were found in the phosphorothioate analogue.     

Extrahelical adenosine stacks into right-handed DNA: solution conformation of the d(C-G-C-A-G-A-G-C-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

This paper reports on features of the three-dimensional structure of the d(C-G-C-A-G-A-G-C-T-C-G-C-G) self-complementary duplex (designated adenosine 13-mer), which contains symmetrical extrahelical adenosines in the interior of the helix. The majority of the protons have been assigned from two-dimensional nuclear Overhauser effect (NOESY) spectra of the adenosine 13-mer in H2O and D2O solution. The measurement of NOESY cross-peak volume integrals as a function of mixing time has yielded a set of 96 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds, which have served as input parameters for a distance geometry analysis of one symmetric half of the adenosine 13-mer duplex. We demonstrate that the extrahelical adenosine stacks into the duplex for all refined structures without disruption of base pairing on either side of the modification site. The distance geometry refinement yields two classes of conformations consistent with distance measurements but which differ in orientation of the stacked extrahelical adenosine at the modification site.     

Three-dimensional structure of proteolytic fragment 163-231 of bacterioopsin determined from nuclear magnetic resonance data in solution.

546 NOESY cross-peak volumes were measured in the two-dimensional NOESY spectrum of proteolytic fragment 163-231 of bacterioopsin in organic solution. These data and 42 detected hydrogen bonds were applied for determining the peptide spatial structure. The fold of the polypeptide chain was determined by local structure analysis, a distance geometry approach and systematic search for energetically allowed side-chain rotamers which are consistent with experimental NOESY cross-peak volumes. The effective rotational correlation time of 6 ns for the molecule was evaluated from optimization of the local structure to meet NOE data and from the dependence on mixing time of the NiH/Ci alpha H cross-peak volumes of the residues in alpha-helical conformation. The resulting structure has two well defined alpha-helical regions, 168-191 and 198-227, with root-mean-square deviation 44 pm and 69 pm, respectively, between the backbone atoms in 14 final energy refined conformations. The alpha-helices correspond to transmembrane segments F and G of bacteriorhodopsin. The segment F contains proline 186, which introduces a kink of about 25 degrees with a disruption of the hydrogen bond with the NH group of the following residue. The segments are connected by a flexible loop region 192-197. Torsion angles chi 1 are unequivocally defined for 62% of side chains in the alpha-helices but half of them differ from electron cryo-microscopy (ECM) model of bacteriorhodopsin, apparently because of the low resolution of ECM. Nevertheless, the F and G segments can be packed as in the ECM model and with side-chain conformations consistent with all NMR data in solution.     

2'-deoxyribonolactone lesion in DNA: refined solution structure determined by nuclear magnetic resonance and molecular modeling

The solution conformation of the DNA duplex d(C1G2C3A4C5L6C7A8C9G10C11).d(G12C13G14T15G16T17G18T19G20C21G22 ) containing the 2'-deoxyribonolactone lesion (L6) in the middle of the sequence has been investigated by NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peak intensities. These distances, along with torsion angles for sugar rings and additional data derived from canonical A- and B-DNA, have been used for structure refinement by restrained molecular dynamics (rMD). Six rMD simulations have been carried out starting from both regular A- and B-DNA forms. The pairwise rms deviations calculated for each refined structure are <1 A, indicating convergence to essentially the same geometry. The accuracy of the rMD structures has been assessed by complete relaxation matrix back-calculation. The average sixth-root residual index (Rx = 0.052 +/- 0.003) indicated that a good fit between experimental and calculated NOESY spectra has been achieved. Detailed analysis revealed a right-handed DNA conformation for the duplex in which both the T17 nucleotide opposite the abasic site and the lactone ring are located inside the helix. No kinking is observed for this molecule, even at the abasic site step. This structure is compared to that of the oligonucleotide with the identical sequence containing the stable tetrahydrofuran abasic site analogue that we reported previously [Coppel, Y., Berthet, N., Coulombeau, C., Coulombeau, Ce., Garcia, J., and Lhomme, J. (1997) Biochemistry 36, 4817-4830].     

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.     

Nuclear magnetic resonance studies of lipid hydration in monomethyldioleoylphosphatidylethanolamine dispersions.

Solid-state proton nuclear magnetic resonance has been used to examine surface hydration in suspensions of monomethyldioleoylphosphatidylethanolamine (MeDOPE). The magic-angle spinning (MAS) 1H spectra for aqueous suspensions of MeDOPE in the L alpha phase exhibited two resonances of roughly equal intensity that could be ascribed to water protons, but both their spin-lattice relaxation times and chemical shifts converged upon conversion to the hexagonal phase. Only a single water peak was observed for analogous samples of dioleoylphosphatidylcholine (DOPC). MAS-assisted two-dimensional nuclear Overhauser effect spectroscopy (NOESY) was conducted for multibilayers of both MeDOPE and DOPC. Through-space interactions were identified between pairs of lipid protons, as expected from their chemical structure. For lamellar suspensions of MeDOPE, positive NOESY cross-peaks were observed between the downfield-shifted water resonance (only) and both CH2N and NH2CH3+ protons of the lipid headgroup. These cross-peaks were not observed in the NOESY spectra of MeDOPE in its hexagonal or cubic phases or for lamellar DOPC reference samples. Taken together, the observation of two water peaks, spin-lattice relaxation behavior, and NOESY connectivities in MeDOPE suspensions support the interpretation that the low-field water peak corresponds to hydrogen-bonded interlamellar water interacting strongly with the lipid. Such a population of water molecules exists in association with MeDOPE in the lamellar phase but not for its inverted phases or for lamellar dispersions of DOPC.     

The differences in the T2 relaxation rates of the protons in the partially-deuteriated and fully protonated sugar residues in a large oligo-DNA ('NMR-window') gives complementary structural information.

Selective incorporation of the stereospecifically deuteriated sugar moieties (> 97 atom % 2H enhancements at H2', H2', H3' and H5'/5' sites, approximately 85 atom % 2H enhancement at H4' and approximately 20 atom % 2H enhancement at H1') in DNA and RNA by the 'NMR-window' approach has been shown to solve the problem of the resonance overlap [refs. 1, 2 & 3]. Such specific deuterium labelling gives much improved resolution and sensitivity of the residual sugar proton (i.e. H1' or H4') vicinal to the deuteriated centers (ref. 3). The T2 relaxation time of the residual protons also increases considerably in the partially-deuteriated (shown by underline) sugar residues in dinucleotides [d(CpG), d(GpC), d(ApT), d(TpA)], trinucleotide r(A2'p5'A2'p5'A) and 20-mer DNA duplex 5'd(C1G2C3-G4C5G6C7G8A9A10T11T12C13G14C15G16C17G18C19G20)(2) 3'. The protons with shorter T2 can be filtered away using a number of different NMR experiments such as ROESY, MINSY or HAL. The NOE intensity of the cross-peaks in these experiments includes only straight pathway from H1' to aromatic proton (i-i and i-i + 1) without any spin-diffusion. The volumes of these NOE cross-peaks could be measured with high accuracy as their intensity is 3 to 4 times larger than the corresponding peaks in the fully protonated residues in the normal NOESY spectra. The structural informations thus obtainable from the residual protons in the partially-deuteriated part of the duplex and the fully protonated part in the 'NMR window' can indeed complement each other.     

Magic-angle spinning NMR studies of molecular organization in multibilayers formed by 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine.

Magic-angle spinning 1H and 13C nuclear magnetic resonance (NMR) have been employed to study 50%-by-weight aqueous dispersions of 1-octadecanoyl-2-decanoyl-sn-glycero-3-phosphocholine (C[18]:C[10]PC) and 1-octadecanoyl-2-d19-decanoyl-PC (C[18]:C[10]PC-d19), mixed-chain phospholipids which can form interdigitated multibilayers. The 1H NMR linewidth for methyl protons of the choline headgroup has been used to monitor the liquid crystalline-to-gel (LC-to-G) phase transition and confirm variations between freezing and melting temperatures. Both 1H and 13C spin-lattice relaxation times indicate unusual restrictions on segmental reorientation at megahertz frequencies for C(18):C(10)PC as compared with symmetric-chain species in the LC state; nevertheless each chemical moiety of the mixed-chain phospholipid exhibits motional behavior that may be classified as liquidlike. Two-dimensional nuclear Overhauser spectroscopy (NOESY) on C(18):C(10)PC and C(18):C(10)PC-d19 reveals cross-peaks between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup, and several experimental and theoretical considerations argue against an interpretation based on spin diffusion. Using NMR relaxation times and NOESY connectivities along with a computational formalism for four-spin systems (Keepers, J. W., and T. L. James. 1984. J. Magn. Reson. 57:404-426), an estimate of 3.5 A is obtained for the average distance between the omega-methyl protons of the C18 chain and the N-methyl protons of the phosphocholine headgroup. This finding is consistent with a degree of interdigitation similar to that proposed for organized assemblies of gel-state phosphatidylcholine molecules with widely disparate acyl-chain lengths (Hui, S. W., and C.-H. Huang. 1986. Biochemistry. 25:1330-1335); however, acyl-chain bendback or other intermolecular interactions may also contribute to the NOESY results. For multibilayers of C(18):C(10)PC in the gel phase, 13C chemical-shift measurements indicate that trans conformers predominate along both acyl chains. 13C Spin-lattice relaxation times confirm the unusual motional restrictions noted in the LC state; nevertheless, 13C and 1H rotating-frame relaxation times indicate that the interdigitated arrangement enhances chain or bilayer motions which occur at mid-kilohertz frequencies.     

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.     

A Renaissance Man: In Memoriam of Jon Widom (1955–2011)

Abstract

Assignment of the ¹H and ³¹P resonances of a decamer DNA duplex, d(CGCTTAAGCG)₂ was determined by two-dimensional COSY, NOESY and ¹H- ³¹P Pure Absorption phase Constant time (PAC) heteronuclear correlation spectroscopy. The solution structure of the decamer was calculated by an iterative hybrid relaxation matrix method combined with NOESY-distance restrained molecular dynamics. The distances from the 2D NOESY spectra were calculated from the relaxation rate matrix which were evaluated from a hybrid NOESY volume matrix comprising elements from the experiment and those calculated from an initial structure. The hybrid matrix-derived distances were then used in a restrained molecular dynamics procedure to obtain a new structure that better approximates the NOESY spectra. The resulting partially refined structure was then used to calculate an improved theoretical NOESY volume matrix which is once again merged with the experimental matrix until refinement is complete. J_H3′-P coupling constants for each of the phosphates of the decamer were obtained from ¹H-³¹P J-resolved selective proton flip 2D spectra. By using a modified Karplus relationship the C4′-C3′-03′-P torsional angles (?) were obtained. Comparison of the ³¹P chemical shifts and J_H3′-P coupling constants of this sequence has allowed a greater insight into the various factors responsible for ³¹P chemical shift variations in oligonucleotides. It also provides an important probe of the sequence-dependent structural variation of the deoxyribose phosphate backbone of DNA in solution. These correlations are consistent with the hypothesis that changes in local helical structure perturb the deoxyribose phosphate backbone. The variation of the ³¹P chemical shift, and the degree of this variation from one base step to the next is proposed as a potential probe of local helical conformation within the DNA double helix. The pattern of calculated ? and ζ torsional angles from the restrained molecular dynamics refinement agrees quite well with the measured J_H3′-P coupling constants. Thus, the local helical parameters determine the length of the phosphodiester backbone which in turn constrains the phosphate in various allowed conformations.     

Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.

The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)