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1.
Although there is a wealth of structural and theoretical data relating to palindromic sequences in genomes, the mechanisms of extrusion of cruciform structures during various biological processes in the presence of intercalating agents are still poorly understood. The current study addresses the effects of temperature and intercalator on cruciform extrusion from plasmids and also considers the effects of divalent metal ions on cruciform extrusion. It presents evidence that the cytotoxic effects of certain DNA binding drugs in vivo occur over concentration ranges corresponding to those that modulate cruciform extrusion in vitro. The results confirm earlier studies showing an inverse relationship between the effects of negative superhelicity and temperature on cruciform extrusion. By extrapolation, divalent metal ions facilitate cruciform extrusion by increasing superhelicity. The results allow the concentrations that preclude cruciform extrusion in DNA to be determined, and these are potentially informative about the relationships among temperature, DNA helical winding, cruciform formation, and intercalation. Overall, we provide new and interesting insights into the potential role of cruciform structures in biology and, by implication, cancer therapy.  相似文献   

2.
Pezza JA  Stopa JD  Brunyak EM  Allen KN  Tolan DR 《Biochemistry》2007,46(45):13010-13018
Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics are critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity and coupling between distal regions on an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase (aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C, distinguished by their unique substrate preferences despite the fact that the structures of the active sites of the three isozymes are nearly identical. While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction of aldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity, those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras were subjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surface patch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9 A. Notably, the coupling energy (DeltaGI) is anticorrelated with respect to the two substrates, fructose 1,6-bisphosphate and fructose 1-phosphate. The difference in coupling energy with respect to these two substrates accounts for approximately 70% of the energy difference for the ratio of kcat/Km for the two substrates between aldolase A and aldolase B. These nonadditive mutational effects between the TSP and DSP provide functional evidence that coupling interactions arising from conformational flexibility during catalysis are a major determinant of substrate specificity.  相似文献   

3.
Cholesterol monomer is incorporated into alkyl sulfate micelles with a unitary free energy of -10.3 kcal/mol. This experimental free energy is in good agreement with that predicted by our previous determination of the hydrophobicity of the sterol suggesting that the partitioning is primarily hydrophobic with little or no contribution to the free energy from head group interactions in this system. The intrinsic hydrophobicity of cholesterol is shown to be insufficient for effective partitioning of the sterol between micelles (or bilayers) and its own self-associated form. This finding strongly supports a model of phospholipid-cholesterol interaction involving significant free energy contributions from head group effects such as alterations in hydrogen bonds or hydration. Since these head group contributions are not observed in the cholesterol-alkyl sulfate system, one concludes that there is a high degree of specificity of interaction between the sterol OH and polar moieties of other amphiphilic molecules.  相似文献   

4.
The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H+ binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pKa values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pKa values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pKa values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking.  相似文献   

5.
Growth hormone conformation and conformational equilibria   总被引:3,自引:0,他引:3  
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6.
Examining solute-induced changes in protein conformational equilibria is a long-standing method for probing the role of water in maintaining protein stability. Interpreting the molecular details governing the solute-induced effects, however, remains controversial. We present experimental and theoretical data for osmolyte-induced changes in the stabilities of the A and N states of yeast iso-1-ferricytochrome c. Using polyol osmolytes of increasing size, we observe that osmolytes alone induce A-state formation from acid-denatured cytochrome c and N state formation from the thermally denatured protein. The stabilities of the A and N states increase linearly with osmolyte concentration. Interestingly, osmolytes stabilize the A state to a greater degree than the N state. To interpret the data, we divide the free energy for the reaction into contributions from nonspecific steric repulsions (excluded volume effects) and from binding interactions. We use scaled particle theory (SPT) to estimate the free energy contributions from steric repulsions, and we estimate the contributions from water-protein and osmolyte-protein binding interactions by comparing the SPT calculations to experimental data. We conclude that excluded volume effects are the primary stabilizing force, with changes in water-protein and solute-protein binding interactions making favorable contributions to stability of the A state and unfavorable contributions to the stability of the N state. The validity of our interpretation is strengthened by analysis of data on osmolyte-induced protein stabilization from the literature, and by comparison with other analyses of solute-induced changes in conformational equilibria.  相似文献   

7.
Rate and thermodynamic parameters have been obtained for the protonation reactions of the aromatic amino acidl-tryptophan using a chemical relaxation technique. These have been compared with values obtained for other aliphatic amino acids. It is shown that the proton rate parameters decrease with increasing pH. This might have biological significance.  相似文献   

8.
9.
Doğan A  Ozel AD  Kiliç E 《Amino acids》2009,36(2):373-379
Knowledge of the protonation constants of small dipeptide is important, interesting and necessary for complete understanding of the physiochemical behavior of dipeptide. In this study, the protonation constants of some aliphatic dipeptides (Gly–Gly, Gly–Val, Gly–Leu, Gly–Thr, Gly–Phe and Gly–Met) were studied in water and ethanol–water mixtures [20% ethanol–80% water, 40% ethanol–60% water, 60% ethanol–40% water, (v/v)] at 25 ± 0.1°C under nitrogen atmosphere and ionic strength at 0.10 mol dm−3 by potentiometry. The constants of the systems were calculated by using BEST computer program, and distribution species diagrams were produced using the SPE computer program. The protonation constants were influenced by changes in solvent composition, and their variations were discussed in terms of solvent and structural properties. The concentration distribution of the various species in ethanol–water mixtures was evaluated.  相似文献   

10.
11.
Structural diversity of N-glycans is essential for specific binding to their receptor proteins. To gain insights into structural and dynamic aspects in atomic detail not normally accessible by experiment, we here perform extensive molecular-dynamics simulations of N-glycans in solution using the replica-exchange method. The simulations show that five distinct conformers exist in solution for the N-glycans with and without bisecting GlcNAc. Importantly, the population sizes of three of the conformers are drastically reduced upon the introduction of bisecting GlcNAc. This is caused by a local hydrogen-bond rearrangement proximal to the bisecting GlcNAc. These simulations show that an N-glycan modification like the bisecting GlcNAc selects a certain “key” (or group of “keys”) within the framework of the “bunch of keys” mechanism. Hence, the range of specific glycan-protein interactions and affinity changes need to be understood in terms of the structural diversity of glycans and the alteration of conformational equilibria by core modification.  相似文献   

12.
13.
14.
Abstract

The effect of dielectric constant of medium on protonation equilibria has been studied by determining protonation constants of ethylenediamine pH metrically in various concentrations (0–60%v/v) of acetoni-trile– and ethylene glycol–water mixtures, at an ionic strength of 0.16mol L?1 and at 303.0 K. MINIQUAD75 computer program has been used for the calculation of protonation constants. Linear and non-linear variations of step-wise protonation constants with reciprocal of dielectric constant of the solvent mixtures have been attributed to the dominance of the electrostatic and non-electrostatic forces, respectively. The trend is explained on the basis of solute–solute and solute–solvent interactions, solvation, proton transfer processes and dielectric constants of the media.  相似文献   

15.
Periole X  Ceruso MA  Mehler EL 《Biochemistry》2004,43(22):6858-6864
Glutamic acid E134 in rhodopsin is part of a highly conserved triad, D(E)RY, located near the cytoplasmic lipid/water interface in transmembrane helix 3 of G protein-coupled receptors (GPCRs). A large body of experimental evidence suggests that the protonation of E134 plays a role in the mechanism of activation of rhodopsin and other GPCRs as well. For E134 to change its protonation state, its pK(a) value must shift from values below physiological pH to higher values. Because of the proximity of the triad to the lipid/water interface, it was hypothesized that a change in solvent around E134 from water to lipid could induce such a shift in pK(a). To test this hypothesis, the pK(a) values of the titratable amino acid residues in rhodopsin have been calculated and the change in solvent around E134 was modeled by shifting the position of the lipid/water interface. The approach used to carry out the pK(a) calculations takes into account the partial immersion of transmembrane proteins in lipid. Qualitative experimental evidence is available for several residues regarding their likely protonation state in rhodopsin at or near physiological pH. Comparison of the calculated pK(a) values with these experimental findings shows good agreement between the two. Notably, glutamic acids E122 and E181 were found to be protonated. The pK(a) values were then calculated for a range of lipid/water interface positions. Although the surrounding solvent of several titratable residues changed from water to lipid in this range, leading to pK(a) shifts in most cases, only for E134 would the shift lead to a change in protonation state at physiological pH. Thus, our results show that the protonation state of E134 is particularly sensitive to its environment. This sensitivity together with the location of E134 near the actual position of the lipid/water interface could be a strategic element in the mechanism of activation of rhodopsin.  相似文献   

16.
Interactions between subunits of the global climate-biosphere system (e.g. atmosphere, ocean, biosphere and cryosphere) often lead to behaviour that is not evident when each subunit is viewed in isolation. This newly evident behaviour is an emergent property of the coupled subsystems. Interactions between thermohaline circulation and climate illustrate one emergent property of coupling ocean and atmospheric circulation. The multiple thermohaline circulation equilibria that result caused abrupt climate changes in the past and may cause abrupt climate changes in the future. Similarly, coupling between the climate system and ecosystem structure and function produces complex behaviour in certain regions. For example, atmosphere-biosphere interactions in the Sahel region of West Africa lead to multiple stable equilibria. Either wet or dry climate equilibria can occur under otherwise identical forcing conditions. The equilibrium reached is dependent on past history (i.e. initial conditions), and relatively small perturbations to either climate or vegetation can cause switching between the two equilibria. Both thermohaline circulation and the climate-vegetation system in the Sahel are prone to abrupt changes that may be irreversible. This complicates the relatively linear view of global changes held in many scientific and policy communities. Emergent properties of coupled socio-natural systems add yet another layer of complexity to the policy debate. As a result, the social and economic consequences of possible global changes are likely to be underestimated in most conventional analyses because these nonlinear, abrupt and irreversible responses are insufficiently considered.  相似文献   

17.
Abstract

The protonation constants of maleic acid and L-asparagine have been studied pH-metrically in various concentrations (0–50% v/v) of acetonitrile–water mixtures maintaining an ionic strength of 0.16 mol L-1 at 300C. The protonation constants have been calculated using the computer program MINIQUAD75 and are selected based on statistical parameters. Linear variation of step-wise protonation constants (log K) with the reciprocal of the dielectric constant of the solvent mixture has been attributed to the dominance of the electrostatic forces.  相似文献   

18.
Ligand binding and internal equilibria in proteins   总被引:13,自引:0,他引:13  
G Weber 《Biochemistry》1972,11(5):864-878
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19.
The light-driven photocycle of rhodopsin begins the photoreceptor cascade that underlies visual response. In a sequence of events, the retinal covalently attached to the rhodopsin protein undergoes a conformational change that communicates local changes to a global conformational change throughout the whole protein. In turn, the large-scale protein change then activates G-proteins and signal amplification throughout the cell. The nature of this change, involving a coupling between a local process and larger changes throughout the protein, may be important for many membrane proteins. In addition, functional work has shown that this coupling occurs with different efficiency in different lipid settings. To begin to understand the nature of the efficiency of this coupling in different lipid settings, we present a molecular dynamics study of rhodopsin in an explicit dioleoyl-phosphatidylcholine bilayer. Our system was simulated for 40 ns and provides insights into the very early events of the visual cascade, before the full transition and activation have occurred. In particular, we see an event near 10 ns that begins with a change in hydrogen bonding near the retinal and that leads through a series of coupled changes to a shift in helical tilt. This type of event, though rare on the molecular dynamics time-scale, could be an important clue to the types of coupling that occur between local and large-scale conformational change in many membrane proteins.  相似文献   

20.
The conformational equilibrium of a decapeptide renin inhibitor (Renin Inhibitory Peptide (RIP), NH-P-H-P-F-H-F-F-V-Y-K-CO2H) in water, methanol and trifluoroethanol has been investigated. The value of a combined spectroscopic approach was apparent, with the need to define conformational states that were mixtures of conformational forms. Similarities between this study and that of the Melanin Concentrating Hormone (MCH) core peptide (5-14) are notable [1]. In water, two beta-turn conformations and an extended form were found to be in equilibrium, with cis/trans isomerism at Pro-3. Extended conformations associated with the P(II) helix and irregular forms were more favoured in aqueous environments. In MeOH and TFE, two beta-turn conformations associated with overlapping sequences and cis/trans isomerism at Pro-3 amide bond were seen to be in equilibrium. 2D ROESY and chemical-exchange cross-peaks were detected by 1H NMR and used to build up detailed models of the interconverting beta-turn conformations of RIP.  相似文献   

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