Nuclear Overhauser Effect Distance Constraints Research Articles

15] Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions

Publisher Summary This chapter describes the application of filamentous bacteriophage as a versatile tool for generating partially ordered solutions of nucleic acids and proteins. The Pfl phage system described in the chapter is an ideal method for obtaining a tunable degree of alignment of RNA and DNA oligomers and some proteins in solution, which then allows the measurement of homonuclear and heteronuclear dipolar coupling interactions. The Pfl filamentous phage system is ideally suited for obtaining a tunable degree of alignment in RNA and DNA molecules and acidic proteins for the observation of dipolar couplings. The phage particles are fully aligned at magnetic fields as low as 300 MHz and are stable indefinitely at a range of temperatures and buffer conditions. These dipolar couplings provide long-range structural information that is presently unavailable by standard solution nuclear magnetic resonance (NMR) techniques; therefore, in the future, dipolar coupling data may prove to be as indispensable as 1 H– 1 H nuclear Overhauser effect (NOE) in the solution structure determinations of macromolecules. This should be especially true for structure determinations of RNA and DNA oligomers where the low density of protons compared to proteins leads to fewer useful NOE distance constraints, and therefore generally less well-defined structures. In addition, the current solution NMR techniques are generally unable to determine global structural features, such as bending in nucleic acids, due because of the local nature of the NOE distance constraints.

Determination of the Stable Microstates of a Peptide from NOE Distance Constraints and Optimization of Atomic Solvation Parameters

A methodology for analyzing nuclear Overhauser effect (NOE) data of interconverting microstates of a peptide has been suggested recently, which is based on pure statistical mechanical considerations. Thus, the most stable microstates and their populations are determined from the free energies. The success of this approach depends on the existence of a reliable potential energy function for the solvated peptide, in which the solvent is treated implicitly. Such a potential is developed here based on the stable structures of the cyclic hexapeptide cyclo(d-Pro1-Phe2-Ala3-Ser4-Phe5-Phe6) in DMSO obtained by Kessler et al. (J. Am. Chem. Soc. 1992, 114, 4805−4818) from NOE distance constraints. This study suggests that two different backbone motifs coexist in equilibrium, one with a βI turn and the other with a βII turn around Ser4-Phe5. We have first reconfirmed these findings by a best-fit analysis applied to a large set of energy-minimized structures generated by our “local torsional deformations” (LTD) method, using the GROMOS force field with and without NOE distance restraints. However, the GROMOS energy EGRO, which excludes solvent interactions was found inappropriate to describe this system because the lowest energy structures representing the βI and βII motifs are ∼15 and 5 kcal/mol above the global energy minimum, respectively. Solvent effects are taken into account through Etot = EGRO + ∑Aiσi, where Ai is the solvent accessible surface area (SASA) of atom i and σi is the atomic solvation parameter (ASP). We optimize the ASPs for DMSO by requiring that the Etot values of βI and βII structures become the lowest globally; this is verified by an extensive application of LTD. The set of ASPs obtained here will be refined in the next work where free energy (rather than energy) considerations will be taken into account. This solvation model, which is relatively easy to handle, requires significantly less computer time than explicit models of solvation and can readily be used in structural analysis of experimental data using GROMOS. The proposed derivation opens the way for the development of similar solvation models for peptides in other solvents. ASPs for proteins in water can be obtained by applying our methodology to surface loops in proteins. Preliminary results for the ASPs, which are slightly different from the present values, were published in a recent Letter (J. Phys. Chem. B 1997, 101, 7368−7370).

The three-dimensional solution structure of the SRC homology domain-2 of the growth factor receptor-bound protein-2.

A set of high-resolution three-dimensional solution structures of the Src homology region-2 (SH2) domain of the growth factor receptor-bound protein-2 was determined using heteronuclear NMR spectroscopy. The NMR data used in this study were collected on a stable monomeric protein solution that was free of protein aggregates and proteolysis. The solution structure was determined based upon a total of 1439 constraints, which included 1326 nuclear Overhauser effect distance constraints, 70 hydrogen bond constraints, and 43 dihedral angle constraints. Distance geometry-simulated annealing calculations followed by energy minimization yielded a family of 18 structures that converged to a root-mean-square deviation of 1.09 A for all backbone atoms and 0.40 A for the backbone atoms of the central beta-sheet. The core structure of the SH2 domain contains an antiparallel beta-sheet flanked by two parallel alpha-helices displaying an overall architecture that is similar to other known SH2 domain structures. This family of NMR structures is compared to the X-ray structure and to another family of NMR solution structures determined under different solution conditions.

Determination of the solution structure of apo calbindin D 9k by NMR spectroscopy

The three-dimensional structure of apo calbindin D9k has been determined using constraints generated from nuclear magnetic resonance spectroscopy. The family of solution structures was calculated using a combination of distance geometry, restrained molecular dynamics, and hybrid relaxation matrix analysis of the nuclear Overhauser effect (NOE) cross-peak intensities. Errors and inconsistencies in the input constraints were identified using complete relaxation matrix analyses based on the results of preliminary structure calculations. The final input data consisted of 994 NOE distance constraints and 122 dihedral constraints, aided by the stereospecific assignment of the resonances from 21 beta-methylene groups and seven isopropyl groups of leucine and valine residues. The resulting family of 33 structures contain no violation of the distance constraints greater than 0.17 A or of the dihedral angle constraints greater than 10 degrees. The structures consist of a well-defined, antiparallel four-helix bundle, with a short anti-parallel beta-interaction between the two unoccupied calcium-binding loops. The root-mean-square deviation from the mean structure of the backbone heavy-atoms for the well-defined helical residues is 0.55 A. The remainder of the ion-binding loops, the linker loop connecting the two sub-domains of the protein, and the N and C termini exhibit considerable disorder between different structures in the ensemble. A comparison with the structure of the (Ca2+)2 state indicates that the largest changes associated with ion-binding occur in the middle of helix IV and in the packing of helix III onto the remainder of the protein. The change in conformation of these helices is associated with a subtle reorganization of many residues in the hydrophobic core, including some side-chains that are up to 15 A from the ion-binding site.

Solution Structure of the Kringle Domain from Urokinase-type Plasminogen Activator

The solution structure of the kringle domain from urokinase-type plasminogen activator (u-PA) has been determined using 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. A total of 35 structures, 20 generated using a distance geometry method prior to simulated annealing and 15 generated using initial random φ, ψ values, have been calculated based on 946 experimental nuclear Overhauser effect distance constraints and 48 dihedral angle constraints. Excluding the N- and C-terminal residues (-1 to 12, 77 to 82) and a number of surface residues (M18, G19, S42, D55 to R60, G67) that are disordered or flexible, the root mean square deviation values from the mean structure are 0·49(±0·14) Å and 0·65(±0·16) Å for the backbone atoms, and 1·03(±0·21) Å and 1·39(±0·24) Å for all heavy atoms, for the two sets of structures, respectively. An extended binding site for anionic polysaccharides such as heparin has been located on a relatively flat facet of the molecule, involving three consecutive arginines, R57, R58 and R60 (there is a deletion at site 59 of the consensus sequence), which form a cationic triad facing the solvent, and two histidines, H37 and H40, at the opposite end of the molecule. Comparison between the u-PA kringle structure and the crystal and NMR solution structures of tissue-type plasminogen activator kringle 2 has shown that the two proteins have similar global folds but demonstrate a number of local differences.

Nuclear Magnetic Resonance Solution Structure of Dendrotoxin K from the Venom of Dendroaspis polylepis polylepis

The solution structure of dendrotoxin K (Toxin K), a protein consisting of one polypeptide chain with 57 residues purified from the venom of the black mamba, Dendroaspis polylepis polylepis, was determined by nuclear magnetic resonance (NMR) spectroscopy. On the basis of virtually complete sequence-specific 1H NMR assignments, including individual assignments for 38 pairs of diastereotopic substituents and side chain amide protons, a total of 818 nuclear Overhauser effect distance constraints and 123 dihedral angle constraints were identified. Using this input, the solution structure of Toxin K was calculated with the program DIANA, and refined by restrained energy minimization with a modified version of the program AMBER. The average root-mean-square deviation (r.m.s.d.) relative to the mean atomic co-ordinates of the 20 conformers selected to represent the solution structure is 0·31 Å for all backbone atoms N, Cα and C′, and 0·90 Å for all heavy atoms of residues 2 to 56. The solution structure of Toxin K is very similar to the solution structure of the basic pancreatic trypsin inhibitor (BPTI) and the X-ray crystal structure of the α-dendrotoxin from Dendroaspis angusticeps (α-DTX), with r.m.s.d. values of 1·31 Å and 0·92 Å, respectively, for the backbone atoms of residues 2 to 56. Some local structural differences between Toxin K and BPTI are directly related to the fact that intermolelcular interactions with two of the four internal molecules of hydration water in BPTI are replaced by intramolecular hydrogen bonds in Toxin K.

Methionyl-tRNA Synthetase Zinc Binding Domain: Three-dimensional Structure and Homology with Rubredoxin and gag Retroviral Proteins

Methionyl-tRNA synthetase from Escherichia coli contains one tightly bound zinc atom per subunit. The region encompassing residues 138 to 163 of this enzyme is responsible for the metal binding. A 28-mer peptide corresponding to these residues was expressed in vivo and shown to contain approximately 1 mol of tightly bound Zn/mol of peptide. In this study, the three-dimensional solution structure of this peptide was solved by means of two-dimensional proton NMR spectroscopy. A total of 133 nuclear Overhauser effect distance constraints and 22 dihedral angle restraints were used for the calculations, using a hybrid distance-geometry-simulated annealing strategy. Excluding the first four residues, the resulting structure is well-defined (r.m.s.d. 0·71 Å for backbone atoms) and composed of a series of four tight turns. The second and the fourth turns are composed of CXXC sequences which are structurally homologous to the NH-S tuns found in the metal binding sites of gag retroviral proteins and rubredoxin. The solution structure of the zinc binding peptide shows significant discrepancies with the crystal structure of methionyl-tRNA synthetase.

Three-dimensional Structure of the Lipoyl domain from Bacillus stearothermophilus Pyruvate Dehydrogenase Multienzyme Complex

The structure of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of nuclear magnetic resonance spectroscopy. A total of 452 nuclear Overhauser effect distance constraints and 76 dihedral angle restraints were employed as the input for the structure calculations, which were performed using a hybrid distance geometry-simulated annealing strategy and the programs DISGEO and X-PLOR. The overall structure of the lipoyl domain (residues 1 to 79 of the dihydrolipoamide acetyltransferase polypeptide chain) is that of a flattened eight-stranded β-barrel folded around a core of well-defined hydrophobic residues. The lipoylation site, lysine 42, is located in the middle of a β-turn, and the N and C-terminal residues of the domain are close together in adjacent β-strands at the opposite end of the molecule. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the C α atoms of residues 15 to 39 and 52 to 76 being almost superimposalble on those of residues 52 to 76 and 15 to 39, respectively (root-mean-square deviation = 1·48 Å). The amino acid residues at key positions in the structure are conserved among all the reported primary structures of lipoyl domains, suggesting that the domains all fold in a similar way.

Sequence-specific 1H n.m.r. assignments and determination of the three-dimensional structure of reduced Escherichia coli glutaredoxin

The determination of the nuclear magnetic resonance structure of reduced E. coli glutaredoxin in aqueous solution is described. Based on nearly complete, sequence-specific resonance assignments, 813 nuclear Overhauser effect distance constraints and 191 dihedral angle constraints were employed as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of reduced glutaredoxin is made up of three helices and four-stranded β-sheet. The first strand of the β-sheet (residues 2 to 7) runs parallel to the second strand (32 to 37) and antiparallel to the third strand (61 to 64), and the sheet is extended in an antiparallel fashion with a fourth strand (67 to 69). The first helix with residues 13 to 28 and the last helix (71 to 83) run parallel to each other on one side of the β-sheet, with their direction opposite to that of the two parallel β-strands, and the helix formed by residues 44 to 53 fills space available due to the twist of the β-sheet and the reduced length of the last two β-strands. The active site Cys11-Pro-Tyr-Cys14 is located after the first β-strand and occupies the latter part of the loop connecting this strand with the first helix.

Sequence-specific 1H n.m.r. assignments and determination of the three-dimensional structure of reduced escherichia coli glutaredoxin

The determination of the nuclear magnetic resonance structure of reduced E. coli glutaredoxin in aqueous solution is described. Based on nearly complete, sequence-specific resonance assignments, 813 nuclear Overhauser effect distance constraints and 191 dihedral angle constraints were employed as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X-PLOR. The molecular architecture of reduced glutaredoxin is made up of three helices and four-stranded β-sheet. The first strand of the β-sheet (residues 2 to 7) runs parallel to the second strand (32 to 37) and antiparallel to the third strand (61 to 64), and the sheet is extended in an antiparallel fashion with a fourth strand (67 to 69). The first helix with residues 13 to 28 and the last helix (71 to 83) run parallel to each other on one side of the β-sheet, with their direction opposite to that of the two parallel β-strands, and the helix formed by residues 44 to 53 fills space available due to the twist of the β-sheet and the reduced length of the last two β-strands. The active site Cys11-Pro-Tyr-Cys14 is located after the first β-strand and occupies the latter part of the loop connecting this strand with the first helix.

Four-dimensional [13C,1H,13C,1H] HMQC-NOE-HMQC NMR spectroscopy: resolving tertiary nuclear Overhauser effect distance constraints in the spectra of larger proteins

Four-dimensional [13C,1H,13C,1H] HMQC-NOE-HMQC NMR spectroscopy: resolving tertiary nuclear Overhauser effect distance constraints in the spectra of larger proteins

NMR studies of the interaction of the antibiotic nogalamycin with the hexadeoxyribonucleotide duplex d(5'-GCATGC)2.

1H resonance assignments in the NMR spectra of the self-complementary hexadeoxyribonucleoside pentaphosphate d(5'-GCATGC)2 and its complex with the antibiotic nogalamycin, together with interproton distance constraints obtained from two-dimensional nuclear Overhauser effect (NOE) spectra, have enabled us to characterize the three-dimensional structure of these species in solution. In the complex described, two drug molecules are bound per duplex, in each of two equivalent binding sites, with full retention of the dyad symmetry. Twenty-eight NOE distance constraints between antibiotic and nucleotide protons define the position and orientation of the bound drug molecule. Nogalamycin intercalates at the 5'-CA and 5'-TG steps with the major axis of the anthracycline chromophore aligned approximately at right angles to the major axes of the base pairs. The nogalose sugar occupies the minor groove of the helix and makes many contacts with the deoxyribose moieties of three nucleotides along one strand of the duplex in the 5'-TGC segment. The charged dimethylamino group and hydroxyl functions of the bicyclic sugar lie in the major groove juxtaposed to the guanine base, the bridging atoms of the bicyclic sugar making contacts with the methyl group of the thymine. Thus the antibiotic is not symmetrically disposed in the intercalation site but is in close contact in both grooves with atoms comprising the 5'-TGC strand. The intercalation cavity is wedge-shaped, the major axes of the base pairs forming the site being tilted with respect to one another. All base-pair hydrogen-bonding interactions are maintained in the complex, and there is no evidence for Hoogsteen pairing. The free duplex adopts a regular right-handed B-type conformation in which all glycosidic bond angles are anti and all sugar puckers lie in the C2'-endo range. In the complex the glycosidic bond angles and the sugar puckers deviate little from those observed for the duplex alone. The presence of two bound nogalamycin molecules substantially slows the "breathing" motions of the base pairs forming the intercalation cavity, and the observation of two downfield-shifted resonances in the 31P NMR spectrum of the complex suggests a pronounced local helix unwinding at the drug binding site. The footprinting data of Fox and Waring [Fox, K.R., & Waring, M.J. (1986) Biochemistry 25, 4349-4356] imply that the highest affinity binding sites of nogalamycin have the sequence 5'-GCA (or 5'-TGC).(ABSTRACT TRUNCATED AT 400 WORDS)