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HU is one of the most abundant proteins in bacterial chromosomes and participates in nucleoid compaction and gene regulation. We report experiments using DNA stretching that study the dependence of DNA condensation by HU on force, salt and HU concentration. Previous experiments at sub-physiological salt levels revealed that low concentrations of HU could compact DNA, whereas larger HU concentrations formed a DNA-stiffening complex. Here we report that this bimodal binding behavior depends sensitively on salt concentration. Only the compaction mode was observed for 150 mM and higher NaCl levels, i.e. for physiological salt concentrations. Similar results were obtained for the more physiological salt K-glutamate. Real-time studies of dissociation kinetics revealed that HU unbound slowly (minutes to hours under the conditions studied) but completely for salt concentrations at or above 100 mM NaCl; the lifetime of HU complexes was observed to increase with the HU concentration at which the complexes were formed, and to decrease with salt concentration. Higher salt levels of 300 mM NaCl completely eliminated observable HU binding to DNA. Finally, we observed that the dissociation kinetics depend on force applied to the DNA: increased applied force in the sub-piconewton range accelerates dissociation, suggesting a mechanism for DNA tension to regulate chromosome structure and gene expression. 相似文献
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Poly(dI-dC).poly(dI-dC) was studied using vibrational circular dichroism and IR spectroscopy in both the base deformation C = O and symmetric PO2- stretching regions. VCD spectra of this duplex under low salt conditions are consistent with its having a B-form structure. Addition of 5 M NaCl leads to relatively uniform VCD intensity loss which is consistent with loss of helical structure rather than formation of an intermediate state between the B and Z forms. This duplex polymer under high salt conditions with added NiCl2 shows aggregation effects, but its IR and VCD spectra have characteristic features of the Z-form DNA conformation. The cooperative change of backbone and base pair structure upon thermal denaturation is indicated by the simultaneous collapse of the VCD at 65 degrees C in both the PO2- and C = O stretching regions. This study further demonstrates that the VCD bandshape of a specific localized nucleic acid vibrational transition can be a useful indicator of the helical handedness. The empirical conformational interpretations are supported by simulated VCD spectra, which are in excellent agreement with the experimental results, based on dipole coupling calculations. 相似文献
5.
Thyrotropin-releasing hormone (TRH) and epidermal growth factor both enhance prolactin synthesis and substrate adhesion (a morphological change called stretching) of GH4 rat pituitary cells. We have examined TRH- and EGF-induced cell stretching using genetic and pharmacologic approaches. We selected and isolated a series of GH4 cell variants nonresponsive to TRH-induced cell stretching (str-). This selection yielded several variants that were nonresponsive to both TRH- and EGF-induced stretching but were still responsive to stretching induced by several other agents (tetradecanoylphorbol acetate [TPA], butyrate, and Neplanocin A). One of the str- variants (a14) was examined in detail. TRH, EGF, and TPA each enhanced prolactin synthesis in a14 cells, indicating that the a14 variant contained functional receptor binding sites for all 3 ligands as well as the capacity to generate those intracellular signals required for enhanced prolactin synthesis. Because the str- variants were isolated without selective pressure for EGF-induced stretching and because the possibility of more than one selectable mutation in all the variants is unlikely, we suggest that TRH and EGF share a common mechanism to induce cell stretching. We next examined whether the str- variants had a defect in a signaling pathway or in the biochemical endpoint for TRH- and EGF-induced cell stretching. A pharmacologic approach was utilized to investigate the biochemical basis for induced cell stretching. A synthetic Arg-Gly-Asp-Ser tetrapeptide (RGDS), specific for fibronectin and vitronectin adhesion receptors, inhibited TRH-, EGF-, and TPA-induced GH4 cell stretching and attachment to fibronectin- and vitronectin-coated dishes. These results suggest that the interaction between fibronectin and/or vitronectin and their receptor(s) may be a biochemical endpoint by which several agonists induced stretching of GH4 cells. Because the str- variant has RGDS-specific binding sites for fibronectin and vitronectin and responds to some agents that induce cell stretching via an RGDS receptor, we conclude that the a14 str- variant has a defect in an intracellular signaling pathway, shared by TRH and EGF, which induces cell stretching. 相似文献
6.
Salt dependence of the elasticity and overstretching transition of single DNA molecules 总被引:9,自引:0,他引:9
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As double-stranded DNA is stretched to its B-form contour length, models of polymer elasticity can describe the dramatic increase in measured force. When the molecule is stretched beyond this contour length, it shows a highly cooperative overstretching transition. We have measured the elasticity and overstretching transition as a function of monovalent salt concentration by stretching single DNA molecules in an optical tweezers apparatus. As the sodium ion concentration was decreased from 1000 to 2.57 mM, the persistence length of DNA increased from 46 to 59 nm, while the elastic stretch modulus remained approximately constant. These results are consistent with the model of Podgornik, et al. (2000, J. Chem. Phys. 113:9343-9350) using an effective DNA length per charge of 0.67 nm. As the monovalent salt concentration was decreased over the same range, the overstretching transition force decreased from 68 to 52 pN. This reduction in force is attributed to a decrease in the stability of the DNA double helix with decreasing salt concentration. Although, as was shown previously, the hydrogen bonds holding DNA strands in a helical conformation break as DNA is overstretched, these data indicate that both DNA strands remain close together during the transition. 相似文献
7.
In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation. 相似文献
8.
Theory of electrostatically regulated binding of T4 gene 32 protein to single- and double-stranded DNA
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Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA binding protein, which is essential for DNA replication, recombination, and repair. In a recent article, we described a new method using single DNA molecule stretching measurements to determine the noncooperative association constants K(ds) to double-stranded DNA for gp32 and *I, a truncated form of gp32. In addition, we developed a single molecule method for measuring K(ss), the association constant of these proteins to single-stranded DNA. We found that in low salt both K(ds) and K(ss) have a very weak salt dependence for gp32, whereas for *I the salt dependence remains strong. In this article we propose a model that explains the salt dependence of gp32 and *I binding to single-stranded nucleic acids. The main feature of this model is the strongly salt-dependent removal of the C-terminal domain of gp32 from its nucleic acid binding site that is in pre-equilibrium to protein binding to both double-stranded and single-stranded nucleic acid. We hypothesize that unbinding of the C-terminal domain is associated with counterion condensation of sodium ions onto this part of gp32, which compensates for sodium ion release from the nucleic acid upon its binding to the protein. This results in the salt-independence of gp32 binding to DNA in low salt. The predictions of our model quantitatively describe the large body of thermodynamic and kinetic data from bulk and single molecule experiments on gp32 and *I binding to single-stranded nucleic acids. 相似文献
9.
In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation. 相似文献
10.
Rupture of a phospholipid bilayer under mechanical stresses is triggered by pore formation in an intact bilayer. To understand the molecular details of the dynamics of pore formation we perform molecular dynamics simulations of a phospholipid bilayer under two different equibiaxial stretching conditions: first, unsteady stretching with various stretching speeds in the range of 0.1-1.0m/s, and second, quasistatic stretching. We analyze (i) patterns of pore formation, (ii) the critical area where a pore forms, (iii) the deformation of the bilayer, and (iv) the apparent breaking force. With stretching, the bilayer deforms anisotropically due to lipid chain packing and water penetrating into the hydrophilic region of the bilayer, and when the area exceeds a critical value, water filled pore structure penetrating the bilayer forms and develops into a large pore, resulting in rupture. For a high stretching speed, small pores (multipore) can temporarily form in a small area. It has been statistically determined that the probability of the multipore formation, the critical areal strain, and the apparent breaking force increase with the stretching speed in the range of 0-50%, 0.8-2.0, and 250-400 pN, respectively. The results qualitatively agree with the experimental and other simulation results, and rationalize the leakage of hemoglobin from erythrocytes in shock wave experiments. 相似文献
11.
Brownian dynamics simulations of probe and self-diffusion in concentrated protein and DNA solutions.
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We have developed a Brownian dynamics algorithm for simulating probe and self-diffusion in concentrated solutions of DNA and protein. In these simulations, proteins are represented as spheres with radii given by their hydrodynamic radii, while DNA is modeled as a wormlike chain of hydrodynamically equivalent spherical frictional elements. The molecular interaction potentials employed by the program allow for intramolecular stretching and bending motions of the DNA chains, short-range Lennard-Jones interactions, and long-range electrostatic interactions. To test the program, we have carried out simulations of bovine serum albumin (BSA) probe diffusion and DNA self-diffusion in solutions of short-chain DNA as a function of both DNA concentration and solution ionic strength. In addition, we report on simulations of BSA self-diffusion as a function of BSA concentration and ionic strength. Based on a comparison to available experimental data, we find that our simulations accurately predict these transport properties under conditions of physiological salt concentration and predict the stronger concentration dependence observed at lower salt concentrations. These results are discussed in light of the nature of the intermolecular interactions in such systems and the approximations and limitations of the simulation algorithm. 相似文献
12.
Temperature-dependent ultraviolet resonance Raman spectroscopy of the premelting state of dA.dT DNA. 总被引:1,自引:1,他引:0
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Poly(dA).poly(dT) and DNA duplex with four or more adenine bases in a row exhibits a broad, solid-state structural premelting transition at about 35 degrees C. The low-temperature structure is correlated with the phenomena of "bent DNA." We have conducted temperature-dependent ultraviolet resonance Raman measurements of the structural transition using poly(dA).poly(dT) at physiological salt conditions, and are able to identify, between the high and low temperature limits, changes in the vibrational frequencies associated with the C4 carbonyl stretching mode in the thymine ring and the N6 scissors mode of the amine in the adenine ring of poly(dA).poly(dT). This work supports the model that the oligo-dA tracts' solid-state structural premelting transition is due to a set of cross-stand bifurcated hydrogen bonds between consecutive dA. dT pairs. 相似文献
13.
We investigate the stretching response of a thick polymer model by means of extensive stochastic simulations. The computational results are synthesized in an analytic expression that characterizes how the force versus elongation curve depends on the polymer structural parameters: its thickness and granularity (spacing of the monomers). The expression is used to analyze experimental data for the stretching of various different types of biopolymers: polypeptides, polysaccharides, and nucleic acids. Besides recovering elastic parameters (such as the persistence length) that are consistent with those obtained from standard entropic models, the approach allows us to extract viable estimates for the polymers diameter and granularity. This shows that the basic structural polymer features have such a profound impact on the elastic behavior that they can be recovered with the sole input of stretching measurements. 相似文献
14.
Cherstvy AG 《European biophysics journal : EBJ》2011,40(1):69-80
We analyze looping of thin charged elastic filaments under applied torques and end forces, using the solution of linear elasticity
theory equations. In application to DNA, we account for its polyelectrolyte character and charge renormalization, calculating
electrostatic energies stored in the loops. We argue that the standard theory of electrostatic persistence is only valid when
the loop’s radius of curvature and close-contact distance are much larger than the Debye screening length. We predict that
larger twist rates are required to trigger looping of charged rods as compared with neutral ones. We then analyze loop shapes
formed on charged filaments of finite length, mimicking DNA looping by proteins with two DNA-binding domains. We find optimal
loop shapes at different salt amounts, minimizing the sum of DNA elastic, DNA electrostatic, and protein elastic energies.
We implement a simple model where intercharge repulsions do not affect the loop shape directly but can choose the energy-optimized
shape from the allowed loop types. At low salt concentrations more open loops are favored due to enhanced repulsion of DNA
charges, consistent with the results of computer simulations on formation of DNA loops by lac repressor. Then, we model the
precise geometry of DNA binding by the lac tetramer and explore loop shapes, varying the confined DNA length and protein opening
angle. The characteristics of complexes obtained, such as the total loop energy, stretching forces required to maintain its
shape, and the reduction of electrostatic energy with increment of salt, are in good agreement with the outcomes of more elaborate
numerical calculations for lac-repressor-induced DNA looping. 相似文献
15.
Das TK Weber RE Dewilde S Wittenberg JB Wittenberg BA Yamauchi K Van Hauwaert ML Moens L Rousseau DL 《Biochemistry》2000,39(46):14330-14340
The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes. 相似文献
16.
We report an unusually high frequency (543 cm(-)(1)) for an Fe-CO stretching mode in the CO complex of Ascaris suum hemoglobin as compared to vertebrate hemoglobins in which the frequency of the Fe-CO mode is much lower. A second Fe-CO stretching mode in Ascaris hemoglobin is observed at 515 cm(-1). We propose that these two Fe-CO stretching modes arise from two protein conformers corresponding to interactions of the heme-bound CO with the B10-tyrosine or the E7-glutamine residues. This postulate is supported by spectra from the B10-Tyr --> Phe mutant in which the 543 cm(-1) line is absent. Thus, a strong polar interaction, such as hydrogen bonding, of the CO with the distal B10 tyrosine residue is the dominant factor that causes this anomalously high frequency. Strong hydrogen bonding between O(2) and distal residues in the oxy complex of Ascaris hemoglobin has been shown to result in a rigid structure, rendering an extremely low oxygen off rate [Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. London B 163, 206-214]. In contrast, the CO off rate in Ascaris hemoglobin is very similar to that in sperm whale myoglobin. The high CO off rate relative to that of O(2) in Ascaris hemoglobin is attributed to a rapid equilibrium between the two conformations of the protein in the CO adduct, with the off rate being determined by the conformer with the higher rate. 相似文献
17.
In Sook Kim Yun Mi Song Tae Hyung Cho Je Yeon Kim Franz E. Weber Soon Jung Hwang 《Journal of biomechanics》2009,42(16):2721-2727
Static stretching is a major type of mechanical stimuli utilized during distraction osteogenesis (DO), a general surgical method for the lengthening of bone. The molecular signals that drive the regenerative process in DO include a variety of cytokines. Among these, bone morphogenic protein (BMP, -2 and -4) has been reported to exhibit strongly enhanced expression following the application of mechanical strain during the distraction phase. We hypothesize that mechanical stretching enhances osteoblast differentiation in DO by means of interaction with BMP-2 induced cytokine stimulation. C2C12 pluripotential myoblasts were exposed to stretching load and the resulting cell proliferation and osteoblast differentiation were then examined. The application of static stretching force resulted in significant cell proliferation at day 3, although with variable intensity according to the magnitude of stretching. A combined treatment of stretching load with BMP-2 stimulation significantly increased alkaline phosphatase (ALP) activity and up-regulated the gene expression of osteogenic markers (ALP, type I collagen, osteopontin, osteocalcin, cbfa1, osterix and dlx5). Results obtained with the combined treatment yielded more activity than just the BMP-2 treatment or stretching alone. These results reveal that specific levels of static stretching force increase cell proliferation and effectively stimulate the osteoblast differentiation of C2C12 cells in conjunction with BMP-2 stimulation, thus indicating a synergistic interaction between mechanical strain and cytokine signaling. 相似文献
18.
We have obtained the resonance Raman spectra of bovine rhodopsin, bathorhodopsin, and isorhodopsin for a series of isotopically labeled retinal chromophores. The specific substitutions are at retinal's protonated Schiff base moiety and include -HC = NH+-, -HC = ND+-, -H13C = NH+-, and -H13C = ND+-. Apart from the doubly labeled retinal, we find that the protonated Schiff base frequency is the same, within experimental error, for both rhodopsin and bathorhodopsin for all the substitutions measured here and elsewhere. We develop a force field that accurately fits the observed ethylenic (C = C) and protonated Schiff base stretching frequencies of rhodopsin and labeled derivatives. Using MINDO/3 quantum mechanical procedures, we investigate the response of this force field, and the ethylenic and Schiff base stretching frequencies, to the placement of charges close to retinal's Schiff base moiety. Specifically, we find that the Schiff base frequency should be measurably affected by a 3.0-4.5-A movement of a negatively charged counterion from the positively charged protonated Schiff base moiety. That there is no experimentally discernible difference in the Schiff base frequency between rhodopsin and bathorhodopsin suggests that models for the efficient conversion of light to chemical energy in the rhodopsin to bathorhodopsin photoconversion based solely on salt bridge separation of the protonated Schiff base and its counterion are probably incorrect. We discuss various alternative models and the role of electrostatics in the rhodopsin to bathorhodopsin primary process. 相似文献
19.
Aguirre JI Plotkin LI Gortazar AR Millan MM O'Brien CA Manolagas SC Bellido T 《The Journal of biological chemistry》2007,282(35):25501-25508
Bone senses and adapts to meet mechanical needs by means of an extensive mechanotransduction network comprising osteocytes (former osteoblasts entrapped in mineral) and their cytoplasmic projections through which osteocytes communicate with osteoblasts and osteoclasts on the bone surface. Mechanical stimulation promotes osteocyte (and osteoblast) survival by activating the extracellular signal-regulated kinases, ERKs. Estrogens have similar effects and, intriguingly, the adaptive response of bone to mechanical forces is defective in mice lacking estrogen receptor (ER) alpha or ERbeta. We report that ERKs are not activated by stretching in osteocytic and osteoblastic cells in which both ERalpha and ERbeta have been knocked out or knocked down and this is reversed partially by transfection of either one of the two human ERs and fully by transfection of both receptors. ERK activation in response to stretching is also recovered by transfecting the ligand-binding domain (E) of either receptor or an ERalpha mutant that does not bind estrogens. Furthermore, mechano-responsiveness is restored by transfecting the Ealpha targeted to the plasma membrane, but not to the nucleus, whereas ERalpha mutants with impaired plasma membrane localization or binding to caveolin-1 fail to confer ERK activation in response to stretching. Lastly, the ER antagonist ICI 182,780 abrogates ERK activation and the anti-apoptotic effect of mechanical stimulation. We conclude that in addition to their role as ligand-dependent mediators of the effects of estrogens, the ERs participate in the transduction of mechanical forces into pro-survival signaling in bone cells, albeit in a ligand-independent manner. 相似文献
20.
A vibrational spectral maker for probing the hydrogen-bonding status of protonated Asp and Glu residues
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Hydrogen bonding is a fundamental element in protein structure and function. Breaking a single hydrogen bond may impair the stability of a protein. We report an infrared vibrational spectral marker for probing the hydrogen-bond number for buried, protonated Asp or Glu residues in proteins. Ab initio computational studies were performed on hydrogen-bonding interactions of a COOH group with a variety of side-chain model compounds of polar and charged amino acids in vacuum using density function theory. For hydrogen-bonding interactions with polar side-chain groups, our results show a strong correlation between the C=O stretching frequency and the hydrogen bond number of a COOH group: approximately 1759-1776 cm(-1) for zero, approximately 1733-1749 cm(-1) for one, and 1703-1710 cm(-1) for two hydrogen bonds. Experimental evidence for this correlation will be discussed. In addition, we show an approximate linear correlation between the C=O stretching frequency and the hydrogen-bond strength. We propose that a two-dimensional infrared spectroscopy, C=O stretching versus O-H stretching, may be employed to identify the specific type of hydrogen-bonding interaction. This vibrational spectral marker for hydrogen-bonding interaction is expected to enhance the power of time-resolved Fourier transform infrared spectroscopy for structural characterization of functionally important intermediates of proteins. 相似文献