Weak NOE Research Articles

Structural and Functional Properties of N‐Methyl‐D‐Aspartate Receptor‐Specific ConRlB

Conantokins are neuroactive peptides found in the venom of marine snails, containing post‐translationally modified γ‐carboxyglutamate residues (γ). The γ residues are integral for their structure, function, and subunit selective antagonism towards N‐Methyl‐D‐Aspartate Receptors (NMDAR). Conantokin RlB (ConRlB) from Conus rolani additionally contains the post‐translationally modified 4‐transhydroxyproline (Hyp; O) residue. The 10 Hyp residue not only changes the γ positioning in the C‐terminal part of ConRlB, but also changes its helicity (62%) in the presence of Mg2+, compared to Mg2+ bound ConG, which is an end‐to‐end α‐helix. The 3D structure of ConRlB, revealed by high‐field 1H NMR, shows disruption in the helix due to 10 O interfering with Mg2+ binding, thus resulting in solvent accessible backbone amide protons. To assess the role of O and the molecular requisites necessary of their unique structural and functional properties, mutant peptides, ConG[10▼O] and ConRlB[ΔKAO▼NQ] were synthesized. Weaker NOEs between residue 9 and 12 of ConG[10▼O], distinguished the nature of the break in the helix due to O compared to ConRlB. Inhibition of intracellular Ca2+ in WT, GluN2A‐/‐, and GluN2B‐/‐ mice cortical neurons by ConG[10▼O] showed increased GluN2A selectivity compared to ConG. ConRlB[ΔKAO▼NQ] showed diminished GluN2B selectivity, in spite of a similar 3D structure to ConG. ConRlB[ΔKAO▼NQ] lost, and ConG[10▼O] retained its ability, to inhibit NMDA‐mediated P‐CREB compared to their respective parent peptides. In conclusion, the O residue changes the structural and functional properties of conantokins.

Rapid protein global fold determination using ultrasparse sampling, high-dynamic range artifact suppression, and time-shared NOESY.

In structural studies of large proteins by NMR, global fold determination plays an increasingly important role in providing a first look at a target's topology and reducing assignment ambiguity in NOESY spectra of fully protonated samples. In this work, we demonstrate the use of ultrasparse sampling, a new data processing algorithm, and a 4-D time-shared NOESY experiment (1) to collect all NOEs in (2)H/(13)C/(15)N-labeled protein samples with selectively protonated amide and ILV methyl groups at high resolution in only four days, and (2) to calculate global folds from this data using fully automated resonance assignment. The new algorithm, SCRUB, incorporates the CLEAN method for iterative artifact removal but applies an additional level of iteration, permitting real signals to be distinguished from noise and allowing nearly all artifacts generated by real signals to be eliminated. In simulations with 1.2% of the data required by Nyquist sampling, SCRUB achieves a dynamic range over 10000:1 (250× better artifact suppression than CLEAN) and completely quantitative reproduction of signal intensities, volumes, and line shapes. Applied to 4-D time-shared NOESY data, SCRUB processing dramatically reduces aliasing noise from strong diagonal signals, enabling the identification of weak NOE crosspeaks with intensities 100× less than those of diagonal signals. Nearly all of the expected peaks for interproton distances under 5 Å were observed. The practical benefit of this method is demonstrated with structure calculations for 23 kDa and 29 kDa test proteins using the automated assignment protocol of CYANA, in which unassigned 4-D time-shared NOESY peak lists produce accurate and well-converged global fold ensembles, whereas 3-D peak lists either fail to converge or produce significantly less accurate folds. The approach presented here succeeds with an order of magnitude less sampling than required by alternative methods for processing sparse 4-D data.

Efficient acquisition of high-resolution 4-D diagonal-suppressed methyl–methyl NOESY for large proteins

The methyl-methyl NOESY experiment plays an important role in determining the global folds of large proteins. Despite the high sensitivity of this experiment, the analysis of methyl-methyl NOEs is frequently hindered by the limited chemical shift dispersion of methyl groups, particularly methyl protons. This makes it difficult to unambiguously assign all of the methyl-methyl NOE crosspeaks using 3-D spectroscopy. The recent development of sparse sampling methods enables highly efficient acquisition of high-resolution 4-D spectra, which provides an excellent solution to resolving the degeneracy of methyl signals. However, many reconstruction algorithms for processing sparsely-sampled NMR data do not provide adequate suppression of aliasing artifacts in the presence of strong NOE diagonal signals. In order to overcome this limitation, we present a 4-D diagonal-suppressed methyl-methyl NOESY experiment specifically optimized for ultrasparse sampling and evaluate it using a deuterated, ILV methyl-protonated sample of the 42 kDa Escherichia coli maltose binding protein (MBP). Suppression of diagonal signals removes the dynamic range barrier of the methyl-methyl NOESY experiment such that residual aliasing artifacts in the CLEAN-reconstructed high-resolution 4-D spectrum can be further reduced. At an ultrasparse sampling rate of less than 1%, we were able to identify and unambiguously assign the vast majority of expected NOE crosspeaks between methyl groups separated by less than 5 Å and to detect very weak NOE crosspeaks from methyl groups that are over 7 Å apart.

Function and Solution Structure of Huwentoxin-X, a Specific Blocker of N-type Calcium Channels, from the Chinese Bird Spider Ornithoctonus huwena

Huwentoxin-X (HWTX-X) is a novel peptide toxin, purified from the venom of the spider Ornithoctonus huwena. It comprises 28 amino acid residues including six cysteine residues as disulfide bridges linked in the pattern of I-IV, II-V, and III-VI. Its cDNA, determined by rapid amplification of 3' and 5' cDNA ends, encodes a 65-residue prepropeptide. HWTX-X shares low sequence homology with omega-conotoxins GVIA and MVIIA, two well known blockers of N-type Ca2+ channels. Nevertheless, whole cell studies indicate that it can block N-type Ca2+ channels in rat dorsal root ganglion cells (IC50 40 nm) and the blockage by HWTX-X is completely reversible. The rank order of specificity for N-type Ca2+ channels is GVIA approximately HWTX-X > MVIIA. In contrast to GVIA and MVIIA, HWTX-X had no detectable effect on the twitch response of rat vas deferens to low frequency electrical stimulation, indicating that HWTX-X has different selectivity for isoforms of N-type Ca2+ channels, compared with GVIA or MVIIA. A comparison of the structures of HWTX-X and GVIA reveals that they not only adopt a common structural motif (inhibitor cystine knot), but also have a similar functional motif, a binding surface formed by the critical residue Tyr, and several basic residues. However, the dissimilarities of their binding surfaces provide some insights into their different selectivities for isoforms of N-type Ca2+ channels.

Extending the limits of the selective 1D NOESY experiment with an improved selective TOCSY edited preparation function

Compared to its 2D counterpart, the selective 1D NOESY experiment offers greatly simplified spectral interpretation and is invaluable to the structure elucidation of small-to-medium sized molecules, although its application is limited to well-resolved resonances only. The doubly selective 1D TOCSY-NOESY experiment allows the 1D NOESY experiment to be extended to resonances within overlapped spectral regions. However, existing methods do not address the critical issue of zero-quantum interference, which leads to severe anti-phase distortions to the line shape of scalar coupled spins and often complicates the identification of weak NOE enhancements. In this communication, we describe an improved selective TOCSY edited preparation (STEP) function and its application to the selective 1D NOESY experiment. The STEP function incorporates a novel zero-quantum filter introduced by Thrippleton and Keeler [Angew. Chem. Int. Ed. 42 (2003) 3938], which permits essentially complete suppression of zero-quantum coherence in a single scan. Residual anti-phase distortions due to spin-state mixing are removed using the double difference methodology reported by Shaka et al. [45th Experimental NMR Conference, Pacific Grove, USA, 2004]. The combined use of these techniques ensures that the final spectra are free of distortions, which is crucial to the reliable detection of weak NOE enhancements. Although employed as an additional preparation period in the example demonstrated here, the STEP function affords a general editing tool for spectral simplification and can be applied to a range of experiments.

Total enantioselectivity in the DNA binding of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [bpm = 2,2'-bipyrimidine; Me2bpy = 4,4'-dimethyl-2,2'-bipyridine

The binding of the three stereoisomers (DeltaDelta-, LambdaLambda- and DeltaLambda-) of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; bpm = 2,2'-bipyrimidine] to a tridecanucleotide containing a single adenine bulge has been studied by 1H NMR spectroscopy. The addition of the DeltaDelta-isomer to d(CCGAGAATTCCGG)2 induced significant chemical shift changes for the base and sugar resonances of the residues at the bulge site (G3A4G5/C11C10), whereas small shifts were observed upon addition of the enantiomeric LambdaLambda-form. NOESY spectra of the tridecanucleotide bound with the DeltaDelta-isomer revealed intermolecular NOE's between the metal complex and the nucleotide residues at the bulge site, while only weak NOE's were observed to terminal residues to the LambdaLambda-form. Competitive binding studies were performed where both enantiomers were simultaneously added to the tridecanucleotide, and for all ratios of the two stereoisomers the DeltaDelta-isomer remained selectively bound at the bulge site with the LambdaLambda-enantiomer localised at the terminal regions of the tridecanucleotide. The meso-diastereoisomer (DeltaLambda) was found to bind to the tridecanucleotide with characteristics intermediate between the DeltaDelta- and LambdaLambda-enantiomers of the rac form. Two distinct sets of metal complex resonances were observed, with one set having essentially the same shift as the free metal complex, whilst the other set of resonances exhibited significant shifts. The NOE data indicated that the meso-diastereoisomer does not bind as selectively as the DeltaDelta-isomer, with NOE's observed to a greater number of nucleotide residues compared to the DeltaDelta-form. This study provides a rare example of total enantioselectivity in the binding of an inert transition metal complex to DNA, produced by the shape recognition of both ruthenium(II) centres.

The Structural Basis of Compstatin Activity Examined by Structure-Function-based Design of Peptide Analogs and NMR

We have previously identified compstatin, a 13-residue cyclic peptide, that inhibits complement activation by binding to C3 and preventing C3 cleavage to C3a and C3b. The structure of compstatin consists of a disulfide bridge and a type I beta-turn located at opposite sides to each other. The disulfide bridge is part of a hydrophobic cluster, and the beta-turn is part of a polar surface. We present the design of compstatin analogs in which we have introduced a series of perturbations in key structural elements of their parent peptide, compstatin. We have examined the consistency of the structures of the designed analogs compared with compstatin using NMR, and we have used the resulting structural information to make structure-complement inhibitory activity correlations. We propose the following. 1) Even in the absence of the disulfide bridge, a linear analog has a propensity for structure formation consistent with a turn of a 3(10)-helix or a beta-turn. 2) The type I beta-turn is a necessary but not a sufficient condition for activity. 3) Our substitutions outside the type I beta-turn of compstatin have altered the turn population but not the turn structure. 4) Flexibility of the beta-turn is essential for activity. 5) The type I beta-turn introduces reversibility and sufficiently separates the two sides of the peptide, whereas the disulfide bridge prevents the termini from drifting apart, thus aiding in the formation of the hydrophobic cluster. 6) The hydrophobic cluster at the linked termini is involved in binding to C3 and activity but alone is not sufficient for activity. 7) beta-Turn residues Gln(5) (Asn(5))-Asp(6)-Trp(7)(Phe(7))-Gly(8) are specific for the turn formation, but only Gln(5)(Asn(5))-Asp(6)-Trp(7)-Gly(8) residues are specific for activity. 8) Trp(7) is likely to be involved in direct interaction with C3, possibly through the formation of a hydrogen bond. Finally we propose a binding model for the C3-compstatin complex.

Automatic assignment of NOESY cross peaks and determination of the protein structure of a new world scorpion neurotoxin using NOAH/DIAMOD.

The 3D NMR structures of the scorpion neurotoxin, CsE-v5, were determined from the same NOESY spectra with NOAH/DIAMOD, an automated assignment and 3D structure calculation software package, and with a conventional manual assignment combined with a distance geometry/simulated annealing (X-PLOR) refinement method. The NOESY assignments and the 3D structures obtained from the two independent methods were compared in detail. The NOAH/DIAMOD program suite uses feedback filtering and self-correcting distance geometry methods to automatically assign NOESY spectra and to calculate the 3D structure of a protein. NOESY cross peaks were automatically picked using a standard software package and combined with 74 manually assigned NOESY peaks to start the NOAH/DIAMOD calculations. After 63 NOAH/DIAMOD cycles, using REDAC procedures in the last 8 cycles, and final FANTOM constrained energy minimization, a bundle of 20 structures with the smallest target functions has a RMSD of 0.81 A for backbone atoms and 1.11 A for all heavy atoms to the mean structure. Despite some missing chemical shifts of side chain protons, 776 (including 74 manually assigned) of 1130 NOE peaks were unambiguously assigned, 150 peaks have more than one possible assignment compatible with the bundle structures, and only 30 peaks could not be assigned within the given chemical shift tolerance ranges in either the D1 or the D2 dimension. The remaining 174, mainly weak NOE peaks were not compatible with the final 20 best bundle structures at the last NOAH/DIAMOD cycle. The automatically determined structures agree well with the structures determined independently using the conventional method and the same NMR spectra, with the mean RMSD in well-defined regions of 0.84 A for bb and 1.48 A for all heavy atoms from residues 2-5, 18-26, 32-36, and 39-45. This study demonstrates the potential of the NOAH/DIAMOD program suite to automatically assign NMR data for proteins and determine their structure.

Structure-Reactivity Studies by NMR Spectroscopy and Molecular Orbital Calculation: Nitration of Polycyclic Aromatic Ketones

Complete assignments of NMR spectra were made for benzanthrones, using 2D correlation spectroscopic techniques of homonuclear H-H COSY and NOESY and heteronuclear CH-COSY and COLOC methods. The nitration positions for 6H-benzo[cd] pyren-6-one (1), 13H-dibenzo[a, de]anthracen-13-one (2), 7H-benzo-[hi]chrysen-7-one (3), 7H-dibenzo[a, kl]anthracen-7-one (4), 5H-benzo[fg]naphthacen-5-one (5) and 7H-benzo[de]anthracen-7-one (6) were determined on the basis of the weak or negative NOE cross peaks, which agreed with the results of the absolute values and phase sensitive 2D NOESY experiment. The nitration positions for the benzanthrones were explained by the appearance of negative or weak NOE cross peaks in the 2D NOESY spectra. Molecular orbital calculations also showed that (1), (2), (4) and (6) have the largest electron densities of the highest occupied molecular orbitals at the carbon atoms where nitration takes place. A correlation can be seen between the 2D NOESY spectra and molecular orbital calculations with respect to the position of nitration.

Backbone--side-chain interactions in serine. Synthesis, crystal structure and solution conformation of a linear model peptide N-Boc-L-Ser-L-Phe-OCH3.

The peptide Boc-Ser-Phe-OCH3 was synthesised by a solution-phase method using the usual workup procedure. The peptide was crystallized from a 70:30 (v/v) methanol-water mixture. The crystals are monoclinic, space group P21 with a = 5.128(2), b = 17.873(2), c = 11.386(2) A, and beta = 98.03(3) degrees. The structure was determined by direct methods and refined by structure factor least-squares procedure. The final R-value for 1499 observed reflections was 0.041. The structure contains one peptide and one solvent water molecule. The peptide adopts a beta-strand-like conformation with phi 1 = -100.3(5), psi 1 = 99.9(5), phi 2 = -122.2(5), psi T2 = -172.5(6) degrees. The Ser side-chain assumes an extended conformation with chi 11 = -177.0(4) degrees. The O gamma H group of serine acts as a proton donor in an intramolecular weak hydrogen bond with (Ser) O'1 [O gamma 1 - H gamma 1 ... O'1 = 3.253(6) A]. The Phe side-chain adopts a staggered conformation with chi 1(2) = -70.9(6), chi 2,1 (2) = 88.4(7) degrees, chi 2,2(2) = -89.2(6) degrees. The water molecule generates a loop through two hydrogen bonds with O gamma 1 [OW ... O gamma 1 = 2.893(5) A] and O'2 [OW ... O'2 = 2.962(7) A] atoms. The unit-translated peptide molecules along the a-axis are held by hydrogen bonds: N1 - H1 ... O2 (chi - 1, y, z) = 2.954(4) A and N2 - H2 ... O'1 (chi + 1, y, z) = 2.897(6) A in a manner similar to those observed in parallel beta-pleated sheet structures. There is an additional interaction involving O gamma 1 and the water molecule [OW ... O gamma 1 (chi + 1, y, z) = 2.789 (4) A]. The strong NOE peak of Ci(H) ... Ni + 1 (H) and a simultaneous weak NOE peak of Ni(H) ... Ni + 1 (H) in the ROESY spectra of two-dimensional NMR in dimethyl sulfoxide indicate a beta-strand-like conformation for the peptide in solution.

Solution conformation of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dC in a DNA duplex.

Combined NMR-molecular mechanics computational studies were undertaken on the C8-deoxyguanosine adduct formed by the carcinogen 1-nitropyrene embedded in the d(C5-[AP]G6-C7).d(G16-C17-G18) sequence context in a 11-mer duplex, with dC opposite the modified deoxyguanosine. The exchangeable and nonexchangeable protons of the aminopyrene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. There was a general broadening of several proton resonances for the three nucleotide d(G16-C17-G18) segment positioned opposite the [AP]dG6 lesion site resulting in weaker NOEs involving these protons in the adduct duplex. The solution conformation of the [AP]dG.dC 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by upper and lower bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs. The modified deoxyguanosine ring of [AP]dG6 is displaced into the major groove and stacks with the major groove edge of dC5 in the adduct duplex. Both carbon and proton chemical shift data for the sugar resonances of the modified deoxyguanosine residue are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue. The dC17 base on the partner strand is displaced from the center of the helix toward the major groove as a consequence of the aminopyrene ring intercalation into the helix. This base-displaced intercalative structure of the [AP]dG.dC 11-mer duplex exhibits several unusually shifted proton resonances which can be accounted for by the ring current contributions of the deoxyguanosinyl and pyrenyl rings of the [AP]dG6 adduct. In summary, intercalation of the aminopyrene moiety is accompanied by displacement of both [AP]dG6 and the partner dC17 into the major groove in the [AP]dG.dC 11-mer duplex.

Synthesis and conformation of constrained peptides with hypoglycaemic activity derived from human growth hormone

The alpha-aminosuccinimide (Asu11) octapeptide analogue of human growth hormone hGH[6-13] (Leu6-Ser-Arg-Leu-Phe-Asu-Asn-Ala13) has been reported [Robson et al. (1990) Biol. Chem. Hoppe Seyler 371, 423-431] to have hypoglycaemic activity whilst the corresponding peptide with Asp at position 11 is inactive. In order to determine whether this change in activity is caused by conformational and/or stereo-electronic effects, the incorporation of two different isomeric gamma-lactam structures at position 11 has been investigated. One lactam structure (i) is of the type developed by Freidinger and coworkers [Freidinger et al. (1982) J. Org. Chem. 47, 102-107], whilst the isomeric gamma-lactam structure (ii) represents a new type of constrained synthon for use in peptide synthesis. The chiral type-ii gamma-lactam was synthesized via a suitably protected desoxodipeptide prepared in several ways from L-aspartic acid. The solution conformations of the [Asu11]- and the [gamma-lactam11]-containing hGH[6-13] peptide analogues were investigated with the aid of two-dimensional NMR (COSY and NOESY) spectroscopy. Conformational similarities were found for these hGH[6-13] peptide analogues. For example, for all peptide analogues studied, weak NOEs were evident between the Phe10 ring protons and protons of the amino acid residues at the C-terminus. Overall, however, the NOESY NMR spectra of the [Asu11]- and the [gamma-lactam11]-containing peptides related to hGH[6-13] suggest the presence of an extended structure in solution with a possible weak type II' beta-turn at position 11. The extent of conformational constraint introduced into these hGH[6-13] peptide analogues by substitution of the Asu11 residue with either isomeric gamma-lactam structure was reflected as differences in their hypoglycaemic activity. In particular, the hGH[6-13] peptide analogue derived from the new chiral type-ii gamma-lactam exhibits both lower activity in intravenous insulin tolerance tests in vivo and weaker NOEs than the isomeric hGH[6-13] peptide analogue derived from the type (i) gamma-lactam structure. The relative change in blood glucose levels from 20 to 90 min for the racemic (R,S)-form of the type-ii gamma-lactam compared to the control values was approximately half that of the (S)-stereoisomer.

1H NMR characterization of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frameshift hotspot of Salmonella typhimurium hisD3052.

The exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was incorporated into 5'-d[ATCGC(PdG)CGGCATG]-3', derived from the hisD3052 gene of Salmonella typhimurium. The modified oligodeoxynucleotide was annealed with the complementary strand 5'-d[CATGCCGCGAT]-3' which contained a CpG deletion. The resulting duplex 5'-d[ATCGC(PdG)CGGCATG]-3'.5'-d[CATGCCGCGAT]-3' required PdG and one adjacent cytosine to be unpaired. Four thymine imino 1H NMR resonances were observed at temperatures below 25 degrees C, which demonstrated formation of a stable duplex with a two-base bulge. PdG was accommodated within the DNA helix, whereas the 3'-neighbor cytosine was poorly stacked and appeared to be extrahelical. The sequential nuclear Overhauser enhancement connectivities between aromatic and H1' protons along the modified strand were interrupted between PdG and the 3'-neighboring unpaired cytosine. On the complementary strand no interruptions were observed. An NOE was observed between the PdG methylene protons H8a,b and the imino proton of the 5'-neighbor base pair. Weaker NOEs were observed between the PdG H8a,b protons and the imino proton from guanine two nucleotides removed in the 3'-direction, and to the amino proton of cytosine located in the complementary strand two nucleotides removed in the 3'-direction. Chemical shift perturbations were also observed for the latter cytosine as compared to the corresponding cytosine in the unmodified fully complementary duplex. These observations provided evidence for a poorly stacked or an extrahelical conformation of this unpaired cytosine. The amino proton resonances of the 3'-neighbor cytosine were not observed, presumably due to increased exchange with solvent. The methylene protons from PdG were shifted upfield relative to the monomer PdG, probably as a result of aromatic ring current shielding, consistent with an intrahelical location. Multimeric derivative oligonucleotides containing the PdG bulge migrated anomalously on nondenaturing polyacrylamide gels, consistent with a structure in which the unpaired nucleotides induced a bend in the DNA.

Folding of peptide fragments comprising the complete sequence of proteins: Models for initiation of protein folding II. Plastocyanin

In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of beta-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the beta-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (phi,psi) space. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the alpha-region of (phi,psi) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.

Multinuclear NMR investigation of zinc(2+) binding to a dodecamer oligodeoxyribonucleotide: insights from carbon-13 NMR spectroscopy

The IH and I3C NMR spectra of the self-complementary dodecamer d(AIT2G,G~G5T6A,CsC9CIOAIIT12)2 were assigned. The dodecamer chemical shifts and NOE effects suggest a normal B-form duplex at 12 OC. However, weak NOE cross peaks from the AH2 signal to the HI' signal of the 3' nucleotide were observed. These weak cross peaks are consistent with some minor sliding in of the propeller-twisted base pairs, particularly at the center of the duplex. At lower concentrations or at higher temperature, a second form of the oligonucelotide was evident. This concentration dependence, the downfield chemical shifts of some 'H resonances, and the observation of an imino signal at 11 ppm strongly imply that this minor form is a hairpin. Addition of Zn2+ greatly disfavored this hairpin form. At lower oligonucleotide concentration, Zn2+ was twice as effective as Mg2+ in favoring the duplex form. The imino 'H spectra established that GC base pairing is not disrupted in the duplex form, even at high levels of added Zn2+. At higher oligonucleotide concentration, when the duplex was converted to the random coil form by temperature elevation, no hairpin form was detected in the presence of Zn2+, whereas such a form was clearly present, particularly at 42 OC, in the absence of Zn2+. At low temperature, when Zn2+ was added, the H8 signals at G4 and, to a lesser extent, G, shifted downfield with little effect on the G5H8 or any AH8 signals. This result suggests a preferential binding to N7 of G4. This conclusion was strongly supported by downfield shifts of the G, and G , C8 signals and the absence of appreciable changes in the C8 signals of other purines. In addition, the C5 signals of G4 and G , shifted upfield and the C4 signals broadened. These characteristic changes strongly support coordination to N7 for these two G residues. However, no significant changes in NOE cross-peak intensities, the C3' I3C signals, or the ,lP NMR signals were observed. Therefore, it is clear that GN7-MCrN7 cross-linked or GMG sandwich species are not formed, since the strain in the sugar-phosphate backbone that would be produced by such species would have led to appreciable changes in chemical shift and NOE cross-peak intensity. The selectivity pattern (G, > G 3 > G5, A) is consistent with the expected molecular electrostatic potential on the basis of literature calculations. Thus, although the study of spectroscopically silent labile metal ions such as ZnZ+ is difficult, a multinuclear NMR investigation of the target biomolecule can be quite revealing.

Relayed-NOE experiments in the rotating frame for sequence analysis of peptides

Proton spectra of peptides consist of a set of spin systems for the individual amino acid residues, which are usually identified via their characteristic J-coupling patterns (I) in 2D correlation spectra. However, the examination of a peptide with unknown amino acid sequence or the presence of several amino acids with identical spin systems requires an assignment of each spin system to its position in the peptide chain. The sequence analysis of a peptide makes use of correlations across the peptide bonds between a-protons and the amide protons of the following amino acid, either via heteronuclear long-range couplings to carbonyl carbons (favorably by COLOC (2) or its inverse variant (3)) or directly via NOE effects (I) (NOESY (4)). However, especially in linear peptides the H, resonances are often not sufficiently resolved for an unambiguous assignment. This serious problem can be circumvented by the use of the Relayed-NOESY experiment (5), advantageously with double-quantum filter (6), where the magnetization transfer via NOE between NH’+’ and HL is followed by a transfer through J coupling from HL to NH’ within the same amino acid residue. This results in a cross peak between NH’ and NH’+’ which appears only on one side of the diagonal due to the asymmetry of the mixing sequence (7) (cf. Fig. 1). Unfortunately, many oligopeptides with molecular weights of about 1000 show only very weak NOE effects in high-field spectrometers due to their unfavorable correlation times. Instead of conventional NOE spectra, ROESY (8, 9) experiments can be used to measure NOE effects in the rotating frame (ROE effects), which are almost independent of the correlation time. We describe here the recently proposed (IO) possibility of using spin-lock sequences in Relayed-NOESY experiments for both the NOE and the Jtransfer part of the mixing time. Figure 2 shows the pulse sequences which are discussed in this paper. The DQF-Relayed-NOESY experiment exists in two versions, which differ in the order of NOE transfer and J trandkr. Since the desired cross peaks always show antiphaae s&uctaue in one dime i$ seems to be favoraMe to have antiphase in w2 because of’tlte bd%cs resduhta in that dimension. This can be achieved by placing ~JVrans~&z&eNOEtrans&ass&owninF~ Z?a.Tbecorrespondingsequence wi~theJ~r~~eNOE~(givingrise~bo~inwl)is~f~ omitted in Fig. 2.

Accounting for spin diffusion in the analysis of 2D NOE data

Abstract Methods used in the quantitative analysis and interpretation of two-dimensional nuclear Overhauser enhancement spectra are described. By taking multispin effects, such as spin diffusion, into account in the analysis, weak NOES can be interpreted and used to obtain additional accurate distance constraints. This increases the number and quality of constraints which can be used to define molecular structures from NMR data.