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A Pardi

Publications and source records attributed to A Pardi.

66 records · Page 4Linked to original sources
PubMed1988

Solution structures of the rabbit neutrophil defensin NP-5.

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.

Animals↗
PubMed1988

Determination of DNA structures by NMR and distance geometry techniques: a computer simulation.

Computer simulations have been performed to determine how accurately and precisely structures of DNA oligomers can be generated from distance data obtained from two-dimensional NMR experiments. A hexamer fragment d(CGAATT) of the Dickerson dodecamer [Drew, H.R., Wing, R.M., Takano, T., Broka, C., Tanaha, S., Itakura, K. & Dickerson, R.E. (1981) Proc. Natl. Acad. Sci. USA 78, 2179-2183] was used as the model structure in these simulations. Protons were added to the coordinates of the original x-ray structure, which was then subjected to a regularization procedure to minimize deviations from standard bond lengths and bond angles. The proton-proton distances normally observed in NMR experiments were measured from this regularized target structure and used as input for a distance geometry algorithm. Distance geometry structures were generated from two distance sets, one with essentially exact distances (+/- 0.005 A) and one set with a precision (+/- 0.2 A) that simulates an optimal NMR experiment. The results of these calculations were used to judge how accurately and precisely the following helical parameters could be reproduced from this simulated NMR distance data: helical twist, helical rise, dislocation, roll, tilt, glycosidic angle, delta torsion angle, and pseudorotation angle. These data provide a basis from which to judge the quality of DNA structures produced from real NMR experiments.

Base Sequence↗
PubMed1987

Two-dimensional NMR studies of the antimicrobial peptide NP-5.

Nearly complete proton resonance assignment of the rabbit antimicrobial peptide NP-5 has been made from two-dimensional NMR data taken at a single temperature. The assignment procedure involved acquisition of phase-sensitive double-quantum-filtered correlation spectra, relayed coherence-transfer spectra, total correlation (homonuclear Hartmann-Hahn) spectra, double- and triple-quantum spectra, and nuclear Overhauser effect spectra. The combination of these complementary experiments simplified and accelerated resonance assignment of the peptide. Individual assignments were made at 20 degrees C for all amide and C alpha protons in the peptide, and for all nonlabile side-chain protons on 26 of the 33 amino acid residues in NP-5. Analysis of the proton-proton nuclear Overhauser effect connectivities, the slowly exchanging amide protons, and the proton chemical shifts in NP-5 indicates that the peptide has a stable, ordered structure in solution. These data also indicate that residues 19-29 in NP-5 are involved in an antiparallel beta-sheet that has a hairpin conformation.

Amino Acid Sequence↗
PubMed1984

Calibration of the angular dependence of the amide proton-C alpha proton coupling constants, 3JHN alpha, in a globular protein. Use of 3JHN alpha for identification of helical secondary structure.

The vicinal amide proton-C alpha proton spin-spin coupling constants, JHN alpha, in the globular protein basic pancreatic trypsin inhibitor (BPTI) have been measured using phase-sensitive correlated spectroscopy at high digital resolution. In conjunction with the crystal structure of BPTI, these data were used to calibrate the correlation between 3JHN alpha and the dihedral angle phi. The resulting "BPTI curve" is 3JHN alpha = 6.4 cos2 theta - 1.4 cos theta + 1.9 (theta = [phi - 60 degrees]). It is further shown that measurement of the spin-spin couplings 3JHN alpha presents an independent, reliable method for identification of the location of helical structure in the amino acid sequence of proteins.

Amides↗
PubMed1983

Protein conformation and proton nuclear-magnetic-resonance chemical shifts.

The nuclear magnetic resonance (NMR) chemical shifts of the polypeptide backbone protons in basic pancreatic trypsin inhibitor from bovine organs and the inhibitors E and K from the venom of Dendroaspis polylepis polylepis have been analyzed. Using the corresponding shifts in model peptides, the chemical shifts observed in the proteins were decomposed into random-coil shifts and conformation-dependent shifts. Correlations between contributions to the latter term and the polypeptide conformation were investigated by using the crystal structure of the bovine inhibitor. In addition to the well-known ring-current effects, a correlation was found between chemical shifts of amide and C alpha protons and the length of the hydrogen bonds formed by these protons with nearby oxygen atoms as acceptor groups. There remain sizeable and as yet unexplained residual conformation shifts. Overall, the present treatment provides a satisfactory qualitative explanation for the outstandingly large shifts of backbone hydrogen atoms in these diamagnetic proteins.

Amino Acid Sequence↗
PubMed1983

Kinetics for exchange of the imino protons of the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix in complexes with the antibiotics netropsin and/or actinomycin.

The lifetimes for exchange of the imino protons in the dodecanucleotide d(C-G-C-G-A-A-T-T-C-G-C-G) upon binding of netropsin and/or actinomycin have been measured by proton nuclear magnetic resonance experiments. At high temperature these lifetimes were found to measure the lifetimes for opening of the base pairs in the double helix. Comparison of the opening rates in the dodecamer with those in the complex with netropsin (which binds at the -A-A-T-T- sequence) shows that there is not only a large kinetic stabilization of the A . T base pairs at the binding site but also a significant stabilization of the G . C base pairs adjacent to the netropsin binding site. For the complex with actinomycin, which intercalates at the G-C sites in the double strand, the lifetimes of the base pairs at the binding site increase upon binding of actinomycin, and the A . T base pairs in the central core are slightly kinetically destabilized by the actinomycin binding. The activation energies for exchange of the imino protons were also measured in the complexes and indicate that the mechanism for exchange of the imino protons is individual base-pair opening, where one base pair opens independently of the others. The effects of drug binding on the dynamics of individual base pairs in a double-stranded helix are discussed.

Base Sequence↗
PubMed1982

Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G).

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.

Adenine↗
PubMed1982

Kinetics for exchange of imino protons in deoxyribonucleic acid, ribonucleic acid, and hybrid oligonucleotide helices.

The lifetime for opening of individual base pairs in a DNA (dCA5G + dCT5G), and RNA (rCA5G + rCU5G), and a hybrid DNA-RNA (rCA5G + dCT5G) helix have been measured by proton nuclear magnetic resonance. The lifetimes were obtained by saturation recovery experiments performed on the hydrogen-bonding imino protons of the Watson-Crick base pairs. In these oligonucleotide helices the observed relaxation rates were dominated by exchange with water, with the magnetic spin-lattice relaxation time of the imino protons possibly being important only at the lowest temperatures in the DNA helix. It was shown that three interior base pairs in the DNA heptamer dCA5G + dCT5G were in the open-limited region, which means that these imino protons exchange every time the base pair opens. The lifetime of the terminal G X C base pairs in the DNA helix are much shorter than the interior A X T base pairs. The pH dependence of the terminal base pairs indicated that the ends of the helix open and close many times before exchange of the imino protons with water takes place. The temperature dependence of the lifetimes of the interior A X T imino protons in the DNA helix showed that these protons exchange only when the double helix has dissociated into single strands. Thus, these lifetimes measure the rate for dissociation of the double helix. The activation energy for this process was found to be 47 kcal/mol. Comparison of the lifetimes of the interior protons in the DNA, RNA, and hybrid helices showed that the rates of dissociation of the RNA and hybrid helices are very similar at 5 degrees C, whereas the rate for the DNA helix was approximately 1 order of magnitude smaller than that for the other two helices. The reasons for the differences in the kinetics of the three helices are discussed, as are the general dynamics of oligonucleotide helices in solution.

Base Sequence↗
PubMed1982

DNA conformation, dynamics, and interactions in solution.

The conformation and dynamics of the d(CGCGAATTCGCG) duplex, its analogs containing mismatched base pairs and helix interruptions, and its complexes with actinomycin and Netropsin, bound separately and simultaneously, have been investigated by nuclear magnetic resonance spectroscopy in aqueous solution. Structural information has been deduced from chemical shift and nuclear Overhauser effect parameters, while the kinetics have been probed from line width and saturation recovery experiments on proton and phosphorus markers at the individual base pair level. These studies lead to an improved understanding of the role of nucleic acid sequence on the structure, flexibility, and conformational interconversions in the duplex state. The nuclear magnetic resonance measurements readily identify helix modification and antibiotic binding sites on the nucleic acid and estimate the extent to which the observed conformational and dynamic perturbations are transmitted to adjacent base pair regions.

Base Sequence↗
PubMed1981

Comparative study of ribonucleotide, deoxyribonucleotide, and hybrid oligonucleotide helices by nuclear magnetic resonance.

The nonexchangeable base protons and the hydrogen-bonding NH--N imino protons were used to study the conformations and the helix--coil transitions in the following oligonucleotides: (I) dCT5G + dCA5G, (II) rCU5G + rCA5G, (III) dCT5 G + rCA5G, (IV) rCU5G + dCA5G. The first three mixtures all form stable double-helical structures at 5 degrees C, whereas IV forms a triple strand with an rCU5G:dCA5G 2:1 ratio. The chemical shifts of the imino protons in the double strands indicate that I, II, and III have different conformations in solution. For example, the hydrogen-bonded proton of one of the C.G base pairs is more deshielded (a 0.4-ppm downfield shift) in helix I than in helix II or III. This implies a significant change in helical parameters, such as the winding angle, the distance between base pairs, or overlap of the bases. The coupling constants of the H1' sugar protons show that helix I has 90% 2'-endo sugar conformation, whereas helix III has greater than 85% 3'-endo conformation for the observed sugar rings. The sugar pucker data are consistent with helix I having B-family geometry; III has A-family geometry. The chemical shifts of the nonexchangeable base protons in system I were followed with increasing temperature. The midpoints for the transitions, Tm's, for all the base protons were 28--30 degrees C; this indicates an all-or-none transition.

Magnetic Resonance Spectroscopy↗
PubMedDate not supplied

Incorporating residual dipolar couplings into the NMR solution structure determination of nucleic acids.

NMR solution structures of nucleic acids are generally less well defined than similar-sized proteins. Most NMR structures of nucleic acids are defined only by short-range interactions, such as intrabase-pair or sequential nuclear Overhauser effects (NOEs), and J-coupling constants, and there are no long-range structural data on the tertiary structure. Residual dipolar couplings represent an extremely valuable source of distance and angle information for macromolecules but they average to zero in isotropic solutions. With the recent advent of general methods for partial alignment of macromolecules in solution, residual dipolar couplings are rapidly becoming indispensable constraints for solution NMR structural studies. These residual dipolar couplings give long-range global structural information and thus complement the strictly local structural data obtained from standard NOE and torsion angle constraints. Such global structural data are especially important in nucleic acids due to the more elongated, less-globular structure of many DNAs and RNAs. Here we review recent progress in application of residual dipolar couplings to structural studies of nucleic acids. We also present results showing how refinement procedures affect the final solution structures of nucleic acids.

DNA↗