Determination of a high-quality nuclear magnetic resonance solution structure of the bovine pancreatic trypsin inhibitor and comparison with three crystal structures.

A high-quality three-dimensional structure of the bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution was determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and compared to the three available high-resolution X-ray crystal structures. A newly collected input of 642 distance constraints derived from nuclear Overhauser effects and 115 dihedral angle constraints was used for the structure calculations with the program DIANA, followed by restrained energy minimization with the program AMBER. The BPTI solution structure is represented by a group of 20 conformers with an average root-mean-square deviation (RMSD) relative to the mean solution structure of 0.43 A for backbone atoms and 0.92 A for all heavy atoms of residues 2 to 56. The pairwise RMSD values of the three crystal structures relative to the mean solution structure are 0.76 to 0.85 A for the backbone atoms and 1.24 to 1.33 A for all heavy atoms of residues 2 to 56. Small local differences in backbone atom positions between the solution structure and the X-ray structures near residues 9, 25 to 27, 46 to 48 and 52 to 58, and conformational differences for individual amino acid side-chains were analyzed for possible correlations with intermolecular protein-protein contacts in the crystal lattices, using the pairwise RMSD values among the three crystal structures as a reference.     

Predicting the Structure of the Flavodoxin from Eschericia coli by Homology Modeling,Distance Geometry and Molecular Dynamics

Abstract

As part of our on-going development of a method, based upon distance geometry calculations, for predicting the structures of proteins from the known structures of their homologues, we have predicted the structure of the 176 residue Flavodoxin from Escherichia coli. This prediction was based upon the crystal structures of the homologous Flavodoxins from Anacystis nidulans, Chondrus crispus, Desulfovibrio vulgaris and Clostridium beijerinckii, whose sequence identities with Escherichia coli were 44%, 33%, 23% and 16%, respectively. A total of 13,043 distance constraints among the alpha-carbons of the Escherichia coli structure were derived from the sequence alignments with the known structures, together with 8,893 distance constraints among backbone and sidechain atoms of adjacent residues, 978 between the alpha-carbons and selected atoms of the flavin mononucleotide cofactor, 116 constraints to enforce conserved hydrogen bonds, and 452 constraints on the torsion angles in conserved residues. An ensemble of ten random Escherichia coli structures was computed from these constraints, with an average root mean square coordinate deviation (RMSD) among the alpha carbons of 0.85 Ångstroms (excluding the first 1 and last 6 residues, which have no corresponding residues in any of the homologues and hence were unconstrained); the corresponding average heavy-atom RMSD was 1.60 Å.

Since the distance geometry calculations were performed without hydrogen atoms, protons were added to the resulting structures and these structures embedded in a 50 × 50 × 40 Å solvent box with periodic boundary conditions. They were then subjected to a 20 picosecond dynamical simulated annealing procedure, starting at 300 K and gradually reduced to 10K, in which all the distance and torsion angle constraints were maintained by means of harmonic restraint functions. This was followed up by 1000 iterations of unrestrained conjugate gradients minimization. The goal of this energy refinement procedure was not to drastically modify the structures in an attempt at a priori prediction, but merely to improve upon the predictions obtained from the geometric constraints, particularly with regard to their local backbone and sidechain conformations and their hydrogen bonds. The resulting structures differed from the respective starting structures by an average of 1.52 Å in their heavy atom RMSD's, while the average RMSD among the heavy atoms of residues 2-170 increased slightly to 1.66 Å. We hope these structures will be good enough to enable the phase problem to be solved for the crystallographic data that is now being collected on this protein.     

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Solution structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa as determined by two-dimensional 1H NMR.

The solution structure of Fe(II) cytochrome c551 from Pseudomonas aeruginosa based on 2D 1H NMR data is reported. Two sets of structure calculations were completed with a combination of simulated annealing and distance geometry calculations: one set of 20 structures included the heme-peptide covalent linkages, and one set of 10 structures excluded them. The main-chain atoms were well constrained within the two structural ensembles (1.30 and 1.35 A average RMSD, respectively) except for two regions spanning residues 30-40 and 60-70. The results were essentially the same when global fold comparisons were made between the ensembles with an average RMSD of 1.33 A. In total, 556 constraints were used, including 479 NOEs, 53 volume constraints, and 24 other distances. This report represents the first solution structure determination of a heme protein by 2D 1H NMR and should provide a basis for the application of these techniques to other proteins containing large prosthetic groups or cofactors.     

Solution structure of apamin determined by nuclear magnetic resonance and distance geometry

The solution structure of the bee venom neurotoxin apamin has been determined with a distance geometry program using distance constraints derived from NMR. Twenty embedded structures were generated and refined by using the program DSPACE. After error minimization using both conjugate gradient and dynamics algorithms, six structures had very low residual error. Comparisons of these show that the backbone of the peptide is quite well-defined with the largest rms difference between backbone atoms in these structures of 1.34 A. The side chains have far fewer constraints and show greater variability in their positions. The structure derived here is generally consistent with the qualitative model previously described, with most differences occurring in the loop between the beta-turn (residues 2-5) and the C-terminal alpha-helix (residues 9-17). Comparisons are made with previously derived models from NMR data and other methods.     

The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X-ray structures.

A high-precision solution structure of the elastase inhibitor eglin c was determined by NMR and distance geometry calculations. A large set of 947 nuclear Overhauser (NOE) distance constraints was identified, 417 of which were quantified from two-dimensional NOE spectra at short mixing times. In addition, a large number of homonuclear 1H-1H and heteronuclear 1H-15N vicinal coupling constants were used, and constraints on 42 chi 1 and 38 phi angles were obtained. Structure calculations were carried out using the distance geometry program DG-II. These calculations had a high convergence rate, in that 66 out of 75 calculations converged with maximum residual NOE violations ranging from 0.17 A to 0.47 A. The spread of the structures was characterized with average root mean square deviations () between the structures and a mean structure. To calculate the unbiased toward any single structure, a new procedure was used for structure alignment. A canonical structure was calculated from the mean distances, and all structures were aligned relative to that. Furthermore, an angular order parameter S was defined and used to characterize the spread of structures in torsion angle space. To obtain an accurate estimate of the precision of the structure, the number of calculations was increased until the and the angular order parameters stabilized. This was achieved after approximately 40 calculations. The structure consists of a well-defined core whose backbone deviates from the canonical structure ca. 0.4 A, a disordered N-terminal heptapeptide whose backbone deviates by 0.8-12 A, and a proteinase-binding loop whose backbone deviates up to 3.0 A. Analysis of the angular order parameters and inspection of the structures indicates that a hinge-bending motion of the binding loop may occur in solution. Secondary structures were analyzed by comparison of dihedral angle patterns. The high precision of the structure allows one to identify subtle differences with four crystal structures of eglin c determined in complexes with proteinases.     

An improved solution structure for psi-conotoxin PiiiE

A revised, high-resolution structure of psi-conotoxin Piiie (psi-Piiie), a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR), produced through the use of NMR and molecular modeling calculations is presented. The original structures of psi-Piiie had a relatively high degree of disorder, particularly in the conformation of the disulfide bridges. Our studies utilized (13)C-labeling of selected cysteine residues allowing the resolution of all problems of resonance overlap for the cysteine residues. The improved data were used to produce a new set of structures by a molecular modeling process incorporating relaxation matrix methods for the determination of interproton distance restraints and a combination of distance geometry and simulated annealing for structure generation. The structures produced are very well converged with the RMSD of backbone atom positions of the main body of the peptide improving from 0.73 to 0.13 A. Other indicators of correlation with the experimental data and quality of covalent geometry showed significant improvement in the new structures. The overall conformation of the peptide backbone is similar between the two determinations with the exception of the N-terminus. This difference leads to a significant effect on the predicted distribution of positive charge within psi-Piiie, a property likely to influence interpretation of future mutational studies.     

The NMR solution structure of the pheromone Er-11 from the ciliated protozoan Euplotes raikovi.

The NMR solution structure of the pheromone Er-11, a 39-residue protein from the ciliated protozoan Euplotes raikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 2-38 was 0.30 A. The molecular architecture is dominated by an up-down-up bundle of three short helices with residues 2-9, 12-19, and 22-32, which is closely similar to the previously determined structures of the homologous pheromones Er-1, Er-2, and Er-10. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell-surface receptors.     

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.     

Three-dimensional structure of (1-36)bacterioopsin in methanol-chloroform mixture and SDS micelles determined by 2D 1H-NMR spectroscopy.

Spatial structures of proteolytic segment A (sA) of bacterioopsin of H. halobium (residues 1-36) solubilized in a mixture of methanol-chloroform (1:1), 0.1 M LiClO4 organic mixture, or in perdeuterated sodium dodecyl sulfate (SDS) micelles, were determined by 2D 1H-NMR techniques. 324 and 400 NOESY cross-peak volumes were measured in NOESY spectra of sA in organic mixture and SDS micelles, respectively. The sA spatial structures were 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 and have the right-handed alpha-helical region from Pro8 to Met32 with root mean square deviation (RMSD) of 0.25 A between backbone heavy atoms and fit well with Pro8 to Met32 alpha-helical region in electron cryo-microscopy model of bacteriorhodopsin. The N-terminal region Ala2-Gly6 of sA in organic mixture has a fixed structure of two consecutive gamma-turns as 2 * 2(7)-helix (RMSD of 0.25 A) stabilized by the Thr5 NH...O = C Gln3 and Ile4 NH...O = C Ala2 hydrogen bonds while this region in SDS micelles has disordered structure with RMSD of 1.44 A for backbone heavy atoms. The C-terminal region Gly33-Asp36 of sA is disordered in both milieus. Torsion angles chi 1 of sA were unequivocally determined for 13 (SDS) and 11 (organic mixture) of alpha-helical residues and are identical in both milieus.     

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Distance constraints from two-dimensional NMR cross-relaxation data are used to derive a three-dimensional structure for acyl carrier protein from Escherichia coli. Several approaches to structure determination are explored. The most successful proves to be an approach that combines the early stages of a distance geometry program with energy minimization in the presence of NMR constraints represented as pseudopotentials. Approximately 450 proton to proton distance constraints including 50 long-range constraints were included in these programs. Starting structures were generated at random by the distance geometry program and energies minimized by a molecular mechanics module to give final structures. Seven of the structures were deemed acceptable on the basis of agreement with experimentally determined distances. Root-mean-square deviations from the mean of these structures for backbone atoms range from 2 to 3 A. All structures show three roughly parallel helices with hydrophobic residues facing inward and hydrophilic residues facing outward. A hydrophobic cleft is recognizable and is identified as a likely site for acyl chain binding.     

RNA loop structure prediction via bond scaling and relaxation

We have developed a method for predicting the structure of small RNA loops that can be used to augment already existing RNA modeling techniques. The method requires no input constraints on loop configuration other than end-to-end distance. Initial loop structures are generated by randomizing the torsion angles, beginning at one end of the polynucleotide chain and correlating each successive angle with the previous. The bond lengths of these structures are then scaled to fit within the known end constraints and the equilibrium bond lengths of the potential energy function are scaled accordingly. Through a series of rescaling and minimization steps the structures are allowed to relax to lower energy configurations with standard bond lengths and reduced van der Waals clashes. This algorithm has been tested on the variable loops of yeast tRNA-Asp and yeast tRNA-Phe, as well as the sarcin-ricin tetraloop and the anticodon loop of yeast tRNA-Phe. The results indicate good correlation between potential energy and the loop structure predictions that are closest to the variable loop crystal structures, but poorer correlation for the more isolated stem loops. The number of stacking interactions has proven to be a good objective measure of the best loop predictions. Selecting on the basis of energy and stacking, we obtain two structures with 0.65 and 0.75 Å all-atom rms deviations (RMSD) from the crystal structure for the tRNA-Asp variable loop. The best structure prediction for the tRNA-Phe variable loop has an all-atom RMSD of 2.2 Å and a backbone RMSD of 1.6 Å, with a single base responsible for most of the deviation. For the sarcin-ricin loop from 28S ribosomal RNA, the predicted structure's all-atom RMSD from the nmr structure is 1.0 Å. We obtain a 1.8 Å RMSD structure for the tRNA-Phe anticodon loop. © 1996 John Wiley & Sons, Inc.     

Binding of retinol induces changes in rat cellular retinol-binding protein II conformation and backbone dynamics

The structure and backbone dynamics of rat holo cellular retinol-binding protein II (holo-CRBP II) in solution has been determined by multidimensional NMR. The final structure ensemble was based on 3980 distance and 30 dihedral angle restraints, and was calculated using metric matrix distance geometry with pairwise Gaussian metrization followed by simulated annealing. The average RMS deviation of the backbone atoms for the final 25 structures relative to their mean coordinates is 0.85(+/-0.09) A. Comparison of the solution structure of holo-CRBP II with apo-CRBP II indicates that the protein undergoes conformational changes not previously observed in crystalline CRBP II, affecting residues 28-35 of the helix-turn-helix, residues 37-38 of the subsequent linker, as well as the beta-hairpin C-D, E-F and G-H loops. The bound retinol is completely buried inside the binding cavity and oriented as in the crystal structure. The order parameters derived from the (15)N T(1), T(2) and steady-state NOE parameters show that the backbone dynamics of holo-CRBP II is restricted throughout the polypeptide. The T(2) derived apparent backbone exchange rate and amide (1)H exchange rate both indicate that the microsecond to second timescale conformational exchange occurring in the portal region of the apo form has been suppressed in the holo form.     

Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.     

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 1H NMR spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 11 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.3 A for residues 2-39 and 0.9 +/- 0.2 A for residues 5-37. The corresponding values for all atoms are 1.9 +/- 0.3 and 1.4 +/- 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N- (residues 1-4) and C- (residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.     

NMR structure of lung surfactant peptide SP-B(11-25)

Surfactant protein B (SP-B) is a 79-residue essential component of lung surfactant, the film of lipid and protein lining the alveoli, and is the subject of great interest for its role in lung surfactant replacement therapies. Here we report circular dichroism results and the solution NMR structure of SP-B(11-25) (CRALIKRIQAMIPKG) dissolved in CD(3)OH at 5 degrees C. This is the first report of NMR data related to the protein SP-B, whose structure promises to help elucidate the mechanism of its function. Sequence-specific resonance assignments were made for all observable (1)H NMR signals on the basis of standard 2D NMR methods. Structures were determined by the simulated annealing method using restraints derived from 2D NOESY data. The calculations yielded 17 energy-minimized structures, three of which were subjected to 0.95 ns of restrained dynamics to assess the relevance of the static structures to more realistic dynamic behavior. Our CD and NMR data confirm that this segment is an amphiphilic alpha helix from approximately residue L14 through M21. The backbone heavy-atom RMSD for residues L14 through M21 is 0.09 +/- 0.12 A, and the backbone heavy-atom RMSD for the whole peptide is 0.96 +/- 2.45 A, the difference reflecting fraying at the termini. Aside from the disordered termini, the minimized structures represent dynamic structures well. Structural similarity to the homologous regions of related saposin-like proteins and the importance of the distribution of polar residues about the helix axis are discussed.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues 20-83) of barley serine proteinase inhibitor 2 (BSPI-2) is determined on the basis of 403 interproton distance, 34 phi backbone torsion angle and 26 hydrogen bonding restraints derived from n.m.r. measurements. A total of 11 converged structures were computed using a metric matrix distance geometry algorithm and refined by restrained molecular dynamics. The average rms difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.2 A for the backbone atoms and 2.1 +/- 0.1 A for all atoms. The overall structure, which is almost identical to that found by X-ray crystallography, is disc shaped and consists of a central four component mixed parallel and antiparallel beta-sheet flanked by a 13 residue alpha-helix on one side and the reactive site loop on the other.     

Monomeric solution structure of the prototypical 'C' chemokine lymphotactin

Lymphotactin, the sole identified member of the C class of chemokines, specifically attracts T lymphocytes and natural killer cells. This 93-residue protein lacks 2 of the 4 conserved cysteine residues characteristic of the other 3 classes of chemokines and possesses an extended carboxyl terminus, which is required for chemotactic activity. We have determined the three-dimensional solution structure of recombinant human lymphotactin by NMR spectroscopy. Under the conditions used for the structure determination, lymphotactin was predominantly monomeric; however, pulsed field gradient NMR self-diffusion measurements and analytical ultracentrifugation revealed evidence of dimer formation. Sequence-specific chemical shift assignments were determined through analysis of two- and three-dimensional NMR spectra of (15)N- and (13)C/(15)N-enriched protein samples. Input for the torsion angle dynamics calculations used in determining the structure included 1258 unique NOE-derived distance constraints and 60 dihedral angle constraints obtained from chemical-shift-based searching of a protein conformational database. The ensemble of 20 structures chosen to represent the structure had backbone and heavy atom rms deviations of 0.46 +/- 0.11 and 1.02 +/- 0.14 A, respectively. The results revealed that human lymphotactin adopts the conserved chemokine fold, which is characterized by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. Two regions are dynamically disordered as evidenced by (1)H and (13)C chemical shifts and [(15)N]-(1)H NOEs: residues 1-9 of the amino terminus and residues 69-93 of the C-terminal extension. A functional role for the C-terminal extension, which is unique to lymphotactin, remains to be elucidated.     

Solution structure of the TnAn DNA duplex GCCGTTAACGCG containing the HpaI restriction site.

The solution structure of the self-complementary DNA duplex [d(GCCGTTAACGGC)]2, which contains the HpaI restriction site GTTAAC, has been elucidated by two-dimensional NMR, distance geometry (DG), and NOE back-calculation methods. Initial distance constraints were determined by polynomial fitting the two-spin initial NOE rates; backbone constraints from NOE and J-coupling observations (Kim et al., 1992) were included. RMSDs between initial-distance-refined structures derived from random-embedded DG, A-DNA, and B-DNA starting structures were all in the range 0.5-1.0 A, indicating good convergence properties of the algorithm, regardless of the starting structure. A semiautomatic back-calculation refinement procedure was developed and used to generate more refined structures for which the BKCALC-simulated NOE volumes matched the experimental data. The six final structures refined from various starting structures exhibit very good agreement with the experimental data (R values = 0.18) and converge well to within 0.8-A RMSD differences for the central 8 base pairs. The torsion and pseudorotation phase angles were found to be well determined by the data, and the local helical parameters for each base step converged quite well. The final structures show that the central T6-A7 step is somewhat underwound (twist angle ca. 29 degrees), with a large negative cup and a normal (wide) minor groove width, while the T5-T6 and A7-A8 steps have a partially narrowed minor groove.