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1.
We report an approach to the problem of detecting and characterising intermediates in the unfolding of ribonuclease A. Two distinct properties of the protein are compared at equilibrium within the unfolding transition zone: (1) a physical property of the protein, the absorbance of buried tyrosine residues, and (2) a functional property, the ability to bind the specific ligand, 2'-CMP. A direct comparison of these two properties is made within the pH 5.8 transition zone, and an indirect comparison is made by using the stopped-flow instrument to sample rapidly the equilibrium properties of the pH 2.0 transition. At both pH 2.0 and pH 5.8, the results indicate that there are no intermediates in folding which have the physical properties of the native enzyme but which have lost the ability to bind a specific ligand. 相似文献
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Yolande Alpert Lszl Cser Bla Farag Frantiek Frank Ferenc Mezei Yuriy M. Ostanevich 《Biopolymers》1985,24(9):1769-1784
The dynamic behavior of pig immunoglobulin G in deuterium oxide solutions was investigated by the neutron spin-echo technique. This novel technique makes it possible to study intramolecular motion without introducing probes into the macromolecule. Using neutron spin-echo, the effective diffusion coefficient, Deff, was obtained as a function of the transferred momentum, κ. For interpretation of the experimental data, two models were designed, and computed Deff values were compared with experimental data. The rigid T-shape model was compatible with the experimental data only by assuming an unrealistically high rotational diffusion coefficient, and it was therefore unacceptable. Reasonable agreement with all available experimental data was obtained with a flexible model of immunoglobulin G molecule, which the Fab arms are assumed to wobble around the hinge region within an angle of 50°. 相似文献
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The 3-deoxy-D-mannooctulosonic acid (KDO) region of lipopolysaccharides (LPS) from the heptoseless mutant Salmonella minnesota R595 and inner core and heptoseless mutants derived from Escherichia coli K12 was studied by 13C NMR spectroscopy. A spin-echo spectral editing technique was employed for the selective detection of the quaternary anomeric carbon of ketosidically linked KDO. Only two quaternary carbon resonances attributable to KDO were detected in the anomeric carbon spectral region of each LPS from heptoseless mutants E. coli D31m4 (99.7 and 100.8 ppm) and S. minnesota R595 (100.0 and 100.9 ppm). Integrated signal intensities from fully relaxed normal 13C spectra showed that equivalent molar quantities of KDO and glucosamine (i.e. 2 mol of each) were present in each of these samples. Similarly, only two KDO anomeric carbon resonances were detected in the LPS from the inner core mutants E. coli D21f1 (100.8 and 101.2 ppm) and E. coli D21e7 (100.8 and 101.2 ppm). These data confirm the presence of a KDO disaccharide structure rather than a trisaccharide as determined by others using thiobarbituric acid-based assays. The LPS of E. coli D21 (complete inner core oligosaccharide) exhibited four quaternary anomeric carbon resonances (99.4, 100.7, 101.8, and 102.7 ppm). The unequal intensities of these resonances, however, demonstrated that significant heterogeneity exists with respect to KDO substitution in this LPS. A third KDO moiety present in substoichiometric amounts could be consistent with this observation. However, this possibility could not be distinguished from other modes of substitutional heterogeneity involving only 2 KDO residues. 相似文献
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《Biophysical journal》2022,121(11):2127-2134
Measuring the mechanical properties of single-stranded DNA (ssDNA) is a complex challenge that has been addressed lately by different methods. We measured the persistence length of ring ssDNA using a combination of a special DNA origami structure, a self-avoiding ring polymer simulation model, and nonparametric estimation statistics. The method overcomes the complexities set forth by previously used methods. We designed the DNA origami nano structures and measured the ring ssDNA polymer conformations using atomic force microscopy. We then calculated their radius of gyration, which was used as a fitting parameter for finding the persistence length. As there is no simple formulation for the radius of gyration distribution, we developed a simulation program consisting of a self-avoiding ring polymer to fit the persistence length to the experimental data. ssDNA naturally forms stem-loops, which should be taken into account in fitting a model to the experimental measurement. To overcome that hurdle, we found the possible loops using minimal energy considerations and used them in our fitting procedure of the persistence length. Due to the statistical nature of the loops formation, we calculated the persistence length for different percentages of loops that are formed. In the range of 25–75% loop formation, we found the persistence length to be 1.9–4.4 nm, and for 50% loop formation we get a persistence length of 2.83 ± 0.63 nm. This estimation narrows the previously known persistence length and provides tools for finding the conformations of ssDNA. 相似文献
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Michael W. Clarkson Ming Lei Elan Z. Eisenmesser Wladimir Labeikovsky Alfred Redfield Dorothee Kern 《Journal of biomolecular NMR》2009,45(1-2):217-225
Protein motions on all timescales faster than molecular tumbling are encoded in the spectral density. The dissection of complex protein dynamics is typically performed using relaxation rates determined at high and ultra-high field. Here we expand this range of the spectral density to low fields through field cycling using the nucleocapsid protein of the SARS coronavirus as a model system. The field-cycling approach enables site-specific measurements of R 1 at low fields with the sensitivity and resolution of a high-field magnet. These data, together with high-field relaxation and heteronuclear NOE, provide evidence for correlated rigid-body motions of the entire β-hairpin, and corresponding motions of adjacent loops with a time constant of 0.8 ns (mesodynamics). MD simulations substantiate these findings and provide direct verification of the time scale and collective nature of these motions. 相似文献
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To determine whether the half-times (T1/2) of the DNA repair processes measured by alkaline elution increased in a dose-dependent manner, exponentially growing 9L/Ro rat brain tumor cells were irradiated with doses of 15-50 Gy, and their DNA repair kinetics was measured by alkaline elution. At 15 Gy, the DNA repair kinetics was biphasic with the fast phase having a T1/2 approximately 6 min and the slow phase having a T1/2 approximately 42 min. As the dose was increased to 50 Gy, the fast-phase T1/2 remained at approximately 6 min, but the slow-phase T1/2 increased to approximately 87 min. Although a dose-dependent increase in the T1/2 of the slow phase of DNA repair (saturation) was measured by alkaline elution, both the absolute value of the slow-phase T1/2 and the dependency of the slow-phase T1/2 on dose were less than those measured by alkaline sucrose gradient sedimentation in zonal rotors with slow reorienting gradient capability. Thus these two techniques appear either to depend on different hydrodynamic properties of the DNA or to have different coefficients of dependency for the same hydrodynamic properties of the DNA. The lower sensitivity for detection of the dose dependency of DNA repair makes it unlikely that the alkaline elution technique will be useful for quantitatively relating the shape of mammalian cell survival curves to the doses at which saturation of a DNA repair process occurs. 相似文献
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Ngwa W Geier O Stallmach F Naji L Schiller J Arnold K 《European biophysics journal : EBJ》2002,31(1):73-80
In this study, the pulsed field gradient (PFG) nuclear magnetic resonance (NMR) technique was used for the investigation of (1) concentration and compression effects on cation self-diffusion, and (2) restricted diffusion of cations in cartilage. Since physiologically relevant cations like Na+ are difficult to investigate owing to their very short relaxation times, the cations tetramethylammonium (TMA) and tetraethylammonium (TEA) were employed for diffusion studies in samples of explanted cartilage. Results indicated that the diffusion of monovalent cations shows strong similarities to observations already made in studies of the diffusion of water in cartilage: with increasing compression, i.e. decreasing water content, the diffusion coefficient of the cation decreases concomitantly. The diffusion coefficients also showed a decrease with increasing cation concentrations, basically reflecting the corresponding decrease in the water content. Both results could be explained by the well-established model of Mackie and Meares. This, together with the similarity of the diffusion coefficient D in cartilage relative to free solution (about 50%) for both cations, is consistent with the view that the water content and not the charge is the most important determinant of the intratissue diffusivity of monovalent cations. Diffusion studies with increasing observation times showed strong evidence of restricted diffusion, allowing the estimation of the geometry of barriers within cartilage. 相似文献
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The binding of ethidium bromide to a DNA hairpin (dU(5)-hairpin) was investigated using a novel 2D diffusion-modulated gradient correlation spectroscopy (DMG-COSY) experiment to evaluate the applicability of this technique for studying the binding of drugs to DNA. The DMG-COSY experiment includes a preparation period during which coherent magnetization is attenuated due to molecular self-diffusion. Magnetization then evolves due to scalar coupling during an evolution delay, and is detected using gradient pulses for coherence selection. The time-domain data are processed in an analogous manner as for gradient-selected COSY experiments. The diffusion coefficient for uridine in DMSO solution was determined from the H5-H6 crosspeak intensities for a series of 2D DMG-COSY experiments that differed in the magnitude of the gradient pulses applied during the preparation period of the DMG-COSY experiment. The diffusion coefficient for uridine calculated from the DMG-COSY experiments was identical (within experimental error) to that determined from 1D diffusion experiments (5.24x10(-6) cm(2)/s at 26 degrees C). The diffusion coefficients for ethidium bromide and for the dU(5)-hairpin were first measured separately using the DMG-COSY experiment, and then measured in the putative complex. The diffusion coefficient for free ethidium bromide (4.15x10(-6) cm(2)/s at 26 degrees C) was considerably larger than for the dU(5)-hairpin (1. 60x10(-6) cm(2)/s at 26 degrees C), as expected for the smaller molecule. The diffusion coefficient for ethidium was markedly decreased upon addition of the dU(5)-hairpin, consistent with complex formation (1.22x10(-6) cm(2)/s at 26 degrees C). Complex formation of 1:1 stoichiometry between ethidium and the stem of the dU(5)-hairpin was verified independently by fluorescence spectroscopy. These results demonstrate the utility of the DMG-COSY experiment for investigating the binding of drugs to DNA in aqueous solution. 相似文献
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Using (1)H NMR spectroscopy, the base-pair opening dynamics of an antiparallel foldback DNA triplex and the corresponding duplex has been characterized via catalyzed imino proton exchange. The triplex system was found to be in an equilibrium between a duplex and a triplex form. The exchange rate between the two forms (i.e., the on/off-rate of the third strand) was measured to be 5 s(-1) at 1 degrees C, and the base-pair dynamics of both forms were investigated separately. Both Watson-Crick and reverse Hoogsteen base pairs were found to have base-pair lifetimes in the order of milliseconds. The stability of the Watson-Crick base pairs was, however, substantially increased in the presence of the third strand. In the DNA triplex, the opening dynamics of the reverse Hoogsteen base pairs was significantly faster than the dynamics of the Watson-Crick pairs. We were able to conclude that, for both Watson-Crick and reverse Hoogsteen base pairs, spontaneous and individual opening from within the closed base triplet is the dominating opening pathway. 相似文献
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Pulsed field gradient diffusion constant measurements were used to resolve the ambiguity in determining the conformational states of single-stranded DNA dodecanucleotides (d1s, d4s and d5s). For d1s and d5s, because of the spectral symmetry conventional NMR analyses cannot differentiate whether they are hairpins or homo-duplexes. However, the diffusion constants of these sequences at 300 K are 1.4 times greater than those of the comparison complementary duplexes. This result agrees well with what is expected for Dhairpin>/Dduplex based on classic liquid-phase translational diffusion models and the Einstein–Stokes equation, confirming that d1s and d5s form hairpins. d4s did not show a structured spectral pattern, but its diffusion constant measurement suggests that this sequence may not be a random coil. The DNA sequences studied contain chemically modified backbone linkages and are potential antisense agents for gene regulation. The knowledge of their diffusion constants, in combination with conventional NMR analysis and other biophysical spectroscopic measurements, provides new insights into the relationships of chemical structure and conformational preference of antisense oligonucleotides and their analogs. 相似文献
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Water permeability of polyunsaturated lipid membranes measured by 17O NMR. 总被引:1,自引:0,他引:1
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Diffusion-controlled water permeation across bilayers of polyunsaturated phospholipids was measured by 17O nuclear magnetic resonance. In 100-nm extruded liposomes containing 50 mM MnCl2, water exchange between internal and external solutions was monitored via changes in the linewidth of the 17O water resonance of external water. Liposome size and shape were characterized by light scattering methods and determination of liposome trapped volume. At 25 degrees C, the following water permeability coefficients were determined: 18:0-18:1n-9 PC, 155 +/- 24 microns/s; 18:0-18:3n-3 PC, 330 +/- 88 microns/s; and 18:0-22:6n-3 PC, 412 +/- 91 microns/s. The addition of 1 M ethanol reduced permeability coefficients to 66 +/- 15 microns/s for 18:0-18:1n-9 PC and to 239 +/- 67 microns/s for 18:0-22:6n-3 PC. Furthermore, the addition of 50 mol% 18:1n-9-18:1n-9 PE reduced the water permeability from 122 +/- 21 microns/s for pure 18:1n-9-18:1n-9 PC to 74 +/- 15 microns/s for the mixture. The significant increase in water permeation for membranes with polyunsaturated hydrocarbon chains correlates with looser packing of polyunsaturated lipids at the lipid-water interface and the suggested deeper penetration of water into these bilayers. Ethanol may block water diffusion pathways by occupying points of water entry into bilayers at the interface. The addition of dioleoylphosphatidylethanolamine increases lipid packing density and, consequently, reduces permeation rates. 相似文献
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Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition 总被引:1,自引:0,他引:1
Changes in the 31P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the 31P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA 31P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated 31P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its 31P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion. 相似文献
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The partial collapse of dipolar and chemical shift tensors for peptide NH and for the amide NH at cross-link sites in cell wall peptidoglycan, of intact lyophilized cells of Aerococcus viridans, indicates NH vector root-mean-square fluctuations of 23 degrees. This result is consistent with the local mobility calculated in typical picosecond regime computer simulations of protein dynamics in the solid state. The experimental root-mean-square angular fluctuations for both types of NH vectors increase to 37 degrees for viable wet cells at 10 degrees C. The similarity in mobilities for both general protein and cell wall peptidoglycan suggests that one additional motion in wet cells involves cooperative fluctuations of segments of cell walls, attached proteins, and associated cytoplasmic proteins. 相似文献
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The complex proton spin-echo decay curve was recorded for human serum albumin (SA) solutions with different concentrations in normal and heavy water. The curve included three fast-decaying components for SA, in addition to the slow-decaying component for the water. The total amplitude of these three components roughly corresponded to the number of protons in the SA (with isotopic exchange taken into account); the component ratio remained constant at different concentrations and different temperatures between 4 and 39 degrees. The relatively slow-decaying protein component, which accounted for similar to 10% of the SA protons, was produced by the side chains of the protein. The presence of two other faster-decaying SA components with approximately equal amplitudes indicated that only about half of the remaining protons in the SA macromolecule are incorporated into the comparatively rigid globule, the other half belonging to groups with high conformational lability in aqueous solution. The activation energy for the aqueous component was close to that for pure water, while the activation energies for the protein components were roughly twice as large. 相似文献