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C G Hoogstraten

Publications and source records attributed to C G Hoogstraten.

9 recordsLinked to original sources
PubMed2001

31P NMR probes of chemical dynamics: paramagnetic relaxation enhancement of the (1)H and (31)P NMR resonances of methyl phosphite and methylethyl phosphate anions by selected metal complexes.

Methyl phosphite ((CH(3)O)P(H)(O)(2)(-); MeOPH) and methylethyl phosphate ((CH(3)O)P(OCH(2)CH(3))(O)(2)(-); MEP) are two members of a class of anionic ligands whose (31)P T(2) relaxation rates are remarkably sensitive to paramagnetic metal ions. The temperature dependence of the (31)P NMR line broadenings caused by the Mn(H(2)O)(6)(2+) ion and a water-soluble manganese(III) porphyrin (Mn(III)TMPyP(5+)) indicates that the extent of paramagnetic relaxation enhancement is a measure of the rate at which the anionic probes come into physical contact with the paramagnetic center (i.e., enter the inner coordination shell); that is, piDeltanu(par) = k(assn)[M], where Deltanu(par) is the difference between the line widths of the resonance in paramagnetic and diamagnetic solutions, and k(assn) is the second-order rate constant for association of the phosphorus ligand with the metal, M. Comparison of the (31)P T(1) and T(2) relaxation enhancements shows that rapid T(2) relaxation by the metal ion is caused by scalar interaction with the electronic spin. Relaxation of the phosphorus-bound proton of MeOPH ((1)H-P) by Mn(III)TMPyP(5+) displayed intermediate exchange kinetics over much of the observable temperature range. The field strength dependence of (1)H-P T(2) enhancement and the independence of the (31)P T(2) support these assertions. As in the case of the (31)P T(2), the (1)H-P T(2) relaxation enhancement results from scalar interaction with the electronic spin. The scalar coupling interpretation of the NMR data is supported by a pulsed EPR study of the interactions of Mn(H(2)O)(6)(2+) with the P-deuterated analogue of methyl phosphite, CH(3)OP((2)H)(O)(2)(-). The electron to (31)P and (2)H nuclear scalar coupling constants were found to be 4.6 and 0.10 MHz, respectively. In contrast, the effects of paramagnetic ions on the methoxy and ethoxy (1)H resonances of MeOPH and MEP are weak, and the evidence suggests that relaxation of these nuclei occurs by a dipolar mechanism. The wide variation in the relaxation sensitivities of the (1)H and (31)P nuclei of MeOPH and MEP permits us to study how differences in the strengths of the interactions between an observed nucleus and a paramagnetic center affect NMR T(2) relaxations. We propose that these anion ligand probes may be used to study ligand-exchange reactivities of manganese complexes without requiring variable temperature studies. The (31)P T(2) is determined by chemical association kinetics when the following condition is met: (T(2M,P)/T(2M,H))(Deltanu(P)/Deltanu(HP) - 1) < 0.2 where T(2M,P) and T(2M,H) are the transverse relaxation times of the (31)P and (1)H nuclei when the probe is bound to the metal, and Deltanu(P) and Deltanu(HP) are the paramagnetic line broadenings of the (31)P and (1)H-P nuclei, respectively. We assert that the ratio T(2M,P)/T(2M,H) can be estimated for a general metal complex using the results of EPR and NMR experiments.

Algorithms↗
PubMed2000

Active site dynamics in the lead-dependent ribozyme.

Conformational dynamics are an important property of ribozymes and other RNA molecules but there is currently only limited information on the relationship between dynamics and RNA function. A recent structural study of the lead-dependent ribozyme, known as the leadzyme, showed significant dynamics at the active site and indicated that a structural rearrangement is required for the reaction to proceed from the ground to the transition state. In this work, microsecond-to-millisecond dynamics of the leadzyme are probed by analysis of the power dependence of (13)C NMR relaxation times in the rotating frame (T(1)(rho)). These results revealed a wide range of conformational dynamics for various residues in the leadzyme. For residue A25 in the active site, the power dependence of T(1)(rho) yielded an exchange lifetime similar to that previously measured by line-shape analysis, and provides an important calibration of this T(1)(rho) methodology for probing the dynamics of macromolecules. Strong evidence was also found for a previously suggested dynamic network of hydrogen bonds stabilizing the GAAA tetraloop motif. Within the active site of the leadzyme, internal motions are observed on a wide variety of time scales, suggesting a complex landscape of accessible states, and potential correlations between observed motions and catalytic function are discussed. These results demonstrate that the power dependence of (13)C T(1)(rho) relaxation times provides a valuable method for probing dynamics in nucleic acids.

Adenine Nucleotides↗
PubMed1998

Order, dynamics and metal-binding in the lead-dependent ribozyme.

The in vitro selected lead-dependent ribozyme is among the smallest and simplest of the known catalytic RNA motifs and has a unique metal ion specificity for divalent lead. The conformation and dynamics of this ribozyme are analyzed here by NMR and chemical probing experiments. Complete assignments of the 1H, 13C, and 15N resonances have been made, and the NMR chemical shift changes in the presence of Pb2+, Mg2+ or high concentrations of Na+ show that there is no significant structural change upon addition of either activating (Pb2+) or inhibitory (Mg2+) divalent ions. The 13C NMR relaxation data indicate substantial dynamic fluctuations on various time-scales for active-site residues in this ribozyme. The combination of chemical probing and NMR experiments reveals a picture of the active site for the lead-dependent ribozyme that has both ordered and dynamic features.

Catalytic Domain↗
PubMed1998

NMR solution structure of the lead-dependent ribozyme: evidence for dynamics in RNA catalysis.

The NMR solution structure of a lead-dependent ribozyme, known as the leadzyme, is presented. This ribozyme is among the smallest of the known catalytic RNAs, with an active site consisting of a six-nucleotide asymmetric internal loop. This loop has a roughly double-helical structure, including a protonated adenine-cytosine wobble base-pair, that positions the cytosine base 5' to the cleavage site in a double-helical conformation. The deviations from helical structure consist of two bulged guanosine residues, G7 and G9, where G7 is the residue 3' to the cleavage site. The scissile phosphate group of the leadzyme is not positioned for in-line nucleophilic attack. Therefore, a conformational rearrangement in the active site is required to reach the proposed transition state for this ribozyme. This is similar to previous observations in X-ray studies of the hammerhead ribozyme, and emphasizes the necessity for dynamic structural fluctuations in the catalytic mechanism of small ribozymes. A model for metal-binding in the leadzyme is proposed in which a lead ion binds to a bulged guanine base that is critical for leadzyme function.

Adenosine↗
PubMed1998

Measurement of carbon-phosphorus J coupling constants in RNA using spin-echo difference constant-time HCCH-COSY.

We report a novel NMR technique for the measurement of carbon-phosphorus coupling constants in RNA oligomers. This method, spin-echo difference constant-time HCCH-COSY, takes advantage of the well-dispersed H1' and C1' resonances to analyze couplings involving the more poorly dispersed ribose carbon and phosphorus resonances. The technique was applied to analysis of the 3JC2'P coupling constants related to backbone epsilon torsion angles in a 30-nucleotide lead-dependent ribozyme. 3JC2'P coupling constants were obtained for approximately 90% of the residues in this RNA, which is over twice as many as could be obtained with previous methods.

Carbon↗
PubMed1998

Improved distance analysis in RNA using network-editing techniques for overcoming errors due to spin diffusion.

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determination of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances with greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are applied to a lead-dependent ribozyme (LZ2). We demonstrate the measurement of both aromatic proton-aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton-ribose proton NOEs free of the efficient cross-relaxation within the ribose ring. In LZ2, the accuracy and precision of the resulting distances are significantly improved. We also find that, by allowing the use of longer mixing times with greater sensitivity, the experimental attenuation of spin diffusion in RNA increases the distance range of interactions that can be analyzed. This effect permits measurement of important long-range distances in LZ2 that are not accessible with standard techniques. Thus, these techniques allow the simultaneous optimization of the number, accuracy, and precision of distance constraints used for RNA structure determinations.

Base Sequence↗
PubMed1996

Effects of experimentally achievable improvements in the quality of NMR distance constraints on the accuracy of calculated protein structures.

New methods for collecting cross-relaxation data from proteins and nucleic acids make it possible to improve the accuracy and precision of interproton distance measurements used as input for NMR solution structure determinations. It thus is of interest to determine whether such experimentally achievable improvements in input distance constraints have significant effects on the precision and accuracy of the resulting structures. To answer this question, we have turned to a computational procedure involving the use of data simulated from a known structure, in order to allow unambiguous assessments of accuracy. The approach to improved distances evaluated here is that afforded by magnetization exchange network editing (MENE); MENE pulse sequences break the network of cross-relaxation interactions into regions that are manipulated so as to defeat certain spin-diffusion terms. A target structure was prepared from the X-ray structure of a small protein, turkey ovomucoid third domain (OMTKY3). A normal NOESY spectrum and two varieties of MENE spectra, BD-NOESY and CBD-NOESY, were simulated by means of complete relaxation matrix analysis. These results were used to create different input data sets with the same number of constraints (perfectly accurate distances derived from the target structure, more accurate distances derived from the MENE simulations, and less accurate distances derived from the NOESY simulation), and these, interpreted at different levels of precision, were used as input for solution structure calculations. The results showed that the use of more precise input data measurably improves the local precision and accuracy of calculated structures, but only if the more precise data include the actual target distance. Incorporation of the experimentally achievable, accurate distances with higher precision afforded by the MENE pulse sequences into the set of input distances was found to improve the accuracy of the resulting structures, particularly in terms of side-chain conformation.

Animals↗
PubMed1995

Comparison of the accuracy of protein solution structures derived from conventional and network-edited NOESY data.

Network-editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin-diffusion pathways. Two network-editing approaches, block-decoupled NOESY and complementary-block-decoupled-NOESY, were applied as three-dimensional, heteronuclear-edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two-hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, J Mol Biol 242:203-214) were improved by their replacement by distances derived from network-edited spectra, and distance geometry/simulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a beta-turn and an expansion of the sampled conformation space in the region of the reactive-site loop. The structures calculated from network-editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.

Animals↗