RESUMEN
We have measured the dependencies of both the dissociation rate of specifically bound EcoRI endonuclease and the ratio of non-specific and specific association constants on water activity, salt concentration, and pH in order to distinguish the contributions of these solution components to specific and non-specific binding. For proteins such as EcoRI that locate their specific recognition site efficiently by diffusing along non-specific DNA, the specific site dissociation rate can be separated into two steps: an equilibrium between non-specific and specific binding of the enzyme to DNA, and the dissociation of non-specifically bound protein. We demonstrated previously that the osmotic dependence of the dissociation rate is dominated by the equilibrium between specific and non-specific binding that is independent of the osmolyte nature. The remaining osmotic sensitivity linked to the dissociation of non-specifically bound protein depends significantly on the particular osmolyte used, indicating a change in solute-accessible surface area. In contrast, the dissociation of non-specifically bound enzyme accounts for almost all the pH and salt-dependencies. We observed virtually no pH-dependence of the equilibrium between specific and non-specific binding measured by the competition assay. The observed weak salt-sensitivity of the ratio of specific and non-specific association constants is consistent with an osmotic, rather than electrostatic, action. The seeming lack of a dependence on viscosity suggests the rate-limiting step in dissociation of non-specifically bound protein is a discrete conformational change rather than a general diffusion of the protein away from the DNA.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasa EcoRI/química , Desoxirribonucleasa EcoRI/metabolismo , Escherichia coli/enzimología , Agua/metabolismo , Sitios de Unión , ADN/química , ADN/genética , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Electricidad Estática , Especificidad por Sustrato , Termodinámica , ViscosidadRESUMEN
Water often acts as a critical reactant in cellular reactions. Its role can be detected by modulating water activity with osmotic agents. We describe the principles behind this 'osmotic stress' strategy, and survey the ubiquity of water effects on molecular structures that have aqueous, solute-excluding regions. These effects are seen with single-functioning molecules such as membrane channels and solution enzymes, as well as in the molecular assembly of actin, the organization of DNA and the specificity of protein/DNA interactions.
Asunto(s)
Líquido Intracelular/química , Líquido Intracelular/metabolismo , Presión Osmótica , Agua/metabolismo , Actinas/metabolismo , ADN/química , ADN/metabolismo , Hemoglobinas/metabolismo , Hexoquinasa/metabolismo , Humanos , Canales Iónicos/metabolismo , Unión Proteica , SolucionesRESUMEN
There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.
Asunto(s)
Presión Osmótica , Agua/química , Sitios de Unión , TermodinámicaRESUMEN
We recently showed that a nonspecific complex of the restriction nuclease EcoRI with poly (dI-dC) sequesters significantly more water at the protein-DNA interface than the complex with the specific recognition sequence. The nonspecific complex seems to retain almost a full hydration layer at the interface. We now find that at low osmotic pressures a complex of the restriction nuclease EcoRI with a DNA sequence that differs by only one base pair from the recognition site (a 'star' sequence) sequesters about 70 waters more than the specific one, a value virtually indistinguishable from nonspecific DNA. Unlike complexes with oligo (dI-dC) or with a sequence that differs by two base pairs from the recognition sequence, however, much of the water in the 'star' sequence complex is removed at high osmotic pressures. The energy of removing this water can be calculated simply from the osmotic pressure work done on the complex. The ability to measure not only the changes in water sequestered by DNA-protein complexes for different sequences, but also the work necessary to remove this water is a potentially powerful new tool for coupling inferred structural changes and thermodynamics.
Asunto(s)
Desoxirribonucleasa EcoRI/química , Polidesoxirribonucleótidos/química , Agua/química , Betaína , Equilibrio HidroelectrolíticoRESUMEN
Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.
Asunto(s)
Acanthamoeba/química , Citoesqueleto de Actina/química , Subfragmentos de Miosina/química , Acanthamoeba/genética , Acanthamoeba/ultraestructura , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestructura , Secuencia de Aminoácidos , Animales , Birrefringencia , Magnesio/química , Microscopía Electrónica , Datos de Secuencia Molecular , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/ultraestructura , Mutación Puntual , Estructura Secundaria de Proteína , Relación Estructura-Actividad , UltracentrifugaciónRESUMEN
The forces that govern DNA double helix organization are being finally systematically measured. The non-specific longer-range interactions--such as electrostatic interactions, hydration, and fluctuation forces--that treat DNA as a featureless rod are reasonably well recognized. Recently, specific interactions--such as those controlled by condensing agents or those consequent to helical structure-are beginning to be recognized, quantified and tested.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Composición de Base , ADN/metabolismo , ADN/ultraestructuraRESUMEN
Forces between type I collagen triple helices are studied in solvents of varying hydrogen-bonding ability. The swelling of collagen fibers in reconstituted films is controlled by the concentration of soluble polymers that are excluded from the fibers and that compete osmotically with collagen for available solvent. The interaxial spacing between the triple helices as a function of the polymer concentration is measured by x-ray diffraction. Exponential-like changes in the spacing with increasing osmotic stress, qualitatively similar to the forces previously found in aqueous solution, are also seen in formamide and ethylene glycol. These are solvents that, like water, are capable of forming three-dimensional hydrogen-bond networks. In solvents that either cannot form a network or have a greatly impaired ability to form a hydrogen-bonded network, strikingly different behavior is observed. A hard-wall repulsion is seen with collagen solvated by ethanol, 2-propanol, and N,N-dimethylformamide. The spacing between helices hardly changes with increasing polymer concentration until the stress exceeds some threshold where removal of the solvent becomes energetically favorable. No solvation of collagen is observed in dimethoxyethane. In solvents with an intermediate ability to form hydrogen-bonded networks, methanol, 2-methoxyethanol, or N-methylformamide, the change in spacing with polymer concentration is intermediate between exponential-like and hard-wall. These results provide direct evidence that the exponential repulsion observed between collagen helices at 0-8-A surface separations in water is due to the energetic cost associated with perturbing the hydrogen-bonded network of solvent molecules between the collagen surfaces.
Asunto(s)
Colágeno/química , Estructura Secundaria de Proteína , Animales , Enlace de Hidrógeno , Sustancias Macromoleculares , Polietilenglicoles , Ratas , Solubilidad , Solventes , Tendones , Agua , Difracción de Rayos X/métodosRESUMEN
The free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme's recognition sequence is linearly dependent on the water chemical potential of the solution, set using several very different solutes, ranging from glycine and glycerol to triethylene glycol and sucrose. This osmotic dependence indicates that the nonspecific complex sequesters some 110 waters more than the specific complex with the recognition sequence. The insensitivity of the difference in number of waters released to the solute identity further indicates that this water is sequestered in a space that is sterically inaccessible to solutes, most likely at the protein-DNA interface of the nonspecific complex. Calculations based on the structure of the specific complex suggest that the apposing DNA and protein surfaces in the nonspecific complex retain approximately a full hydration layer of water.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasa EcoRI/metabolismo , Agua , Sitios de Unión , Fenómenos Químicos , Química Física , Electroforesis en Gel de Poliacrilamida , Cinética , Concentración Osmolar , Polidesoxirribonucleótidos/metabolismo , Relación Estructura-ActividadRESUMEN
The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II minifilaments is dependent both on Mg2+ concentration and on the state of phosphorylation of three serine sites at the C-terminal end of the heavy chains. Previous electric birefringence experiments on minifilaments showed a large dependence of signal amplitude on the phosphorylation state and Mg2+ concentration, consistent with large changes in filament flexibility. These observations suggested that minifilament stiffness was important for function. We now report that the binding of nucleotides to dephosphorylated minifilaments at Mg2+ concentrations needed for optimal activity increases the flexibility by about 10-fold, as inferred from the birefringence signal amplitude increase. An increase in flexibility with nucleotide binding is not observed for dephosphorylated minifilaments at lower Mg2+ concentrations or for phosphorylated minifilaments at any Mg2+ concentrations examined. The relaxation times for minifilament rotations that are sensitive to the conformation myosin heads are also observed to depend on phosphorylation, Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg2+ concentration, and nucleotide binding. These latter experiments indicate that the actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin II correlates with both changes in myosin head conformation and the ability of minifilaments to cycle between stiff and flexible conformations coupled to nucleotide binding and release.
Asunto(s)
Acanthamoeba/metabolismo , Nucleótidos de Adenina/farmacología , Miosinas/metabolismo , Animales , ATPasa de Ca(2+) y Mg(2+)/metabolismo , Miosinas/química , Fosforilación , Unión Proteica , Conformación Proteica , Transducción de SeñalRESUMEN
By equilibrating condensed DNA arrays against reservoirs of known osmotic stress and examining them with several structural probes, it has been possible to achieve a detailed thermodynamic and structural characterization of the change between two distinct regions on the liquid-crystalline phase diagram: (i) a higher density hexagonally packed region with long-range bond orientational order in the plane perpendicular to the average molecular direction and (ii) a lower density cholesteric region with fluid-like positional order. X-ray scattering on highly ordered DNA arrays at high density and with the helical axis oriented parallel to the incoming beam showed a sixfold azimuthal modulation of the first-order diffraction peak that reflects the macroscopic bond-orientational order. Transition to the less-dense cholesteric phase through osmotically controlled swelling shows the loss of this bond orientational order, which had been expected from the change in optical birefringence patterns and which is consistent with a rapid onset of molecular positional disorder. This change in order was previously inferred from intermolecular force measurements and is now confirmed by 31P NMR. Controlled reversible swelling and compaction under osmotic stress, spanning a range of densities between approximately 120 mg/ml to approximately 600 mg/ml, allow measurement of the free-energy changes throughout each phase and at the phase transition, essential information for theories of liquid-crystalline states.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Espectroscopía de Resonancia Magnética , Presión Osmótica , Dispersión de Radiación , Termodinámica , Difracción de Rayos X , Rayos XRESUMEN
Simultaneous measurements on the packing and energetics of high-density liquid crystalline DNA phases show that the crowding of long DNA polyelectrolytes at ever increasing concentrations is accomplished through straightening of the random coils that the double helix assumes in dilute solution. X-ray scattering by ordered phases reveals that the local straightening of the molecules is also accompanied by their progressive immobilization and confinement within the molecular 'cages' created by neighboring molecules. These effects can be clearly observed through the measured energies of DNA packing under osmotic stress and through the changes in structural and dynamic characteristics of X-ray scattering from DNA in ordered arrays at different concentrations. The character of the confinement of large DNA motions for a wide range of DNA concentrations is dominated by the soft potentials of direct interaction. We do not see the power-law variation of energy vs. volume expected from space-filling fluctuations of molecules that enjoy no interaction except the hard clash of steric repulsion. Rather, in highly concentrated DNA mesophases we see a crowding of molecules through electrostatic or hydration repulsion that confines their movements and positions. This view is based on directly measured packing energies as well as on concurrently measured structural parameters while the DNA double helices are condensed under an externally applied osmotic pressure.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Presión Osmótica , Dispersión de Radiación , Rayos XRESUMEN
Several neutral solutes, ranging in size from methanol to a tetrasaccharide, stachyose, are shown to stabilize the left-handed Z form of the methylated polynucleotide poly(dG-m5dC). The action of these solutes is consistent with an osmotic stress, that is, with their effect on water chemical potentials coupled to a difference in the number of associated water molecules between the B and Z conformations. The apparent difference in hydration between the two forms is, however, dependent on the particular solute used to probe the reaction. The effect of solutes is not consistent either with a direct binding of solute or with an indirect effect on electrostatics or ion binding through changes in the solution dielectric constant. The interplay of NaCl and neutral solute in modulating the B-Z transition suggests that salt also could be stabilizing the Z form through an osmotic stress.
Asunto(s)
Polinucleótidos/química , Modelos Moleculares , Conformación de Ácido Nucleico , Presión OsmóticaRESUMEN
We find that netropsin and netropsin analogue protect DNA from EcorI restriction nuclease cleavage by inhibiting the binding of EcoRI to its recognition site. The drug -- EcoRI competitive binding constants measured by a electrophoretic gel mobility shift assay are in excellent agreement with the nuclease protection results for the netropsin analogue and in reasonable agreement for netropsin itself. Crystal structures of complexes show that netropsin and EcoRI recognize different regions of the DNA helix and would not be expected to compete for binding to the restriction nuclease site. The large distortions in DNA structure caused by EcoRI binding are most likely responsible for an indirect structural competition with netropsin binding. The structural change in the netropsin binding region induced by EcoRI binding to its region essentially prevents drug association. Given the reciprocal nature of competition, binding of netropsin to a minimally perturbed structure then also makes the association of EcoRI energetically more costly. Since many sequence specific DNA binding proteins significantly bend or distort the DNA helix, drugs that compete indirectly can be as effective as drugs that act through a direct steric inhibition.
Asunto(s)
Antibacterianos/química , Antivirales/química , ADN/química , Netropsina/química , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Desoxirribonucleasa EcoRI , Datos de Secuencia Molecular , Estructura Molecular , Netropsina/análogos & derivadosRESUMEN
The binding of a netropsin analogue to random sequence DNA, monitored by CD, is seen dependent on the concentration of neutral solutes. The binding free energy decreases linearly with solute osmolal concentration and the magnitude of the effect is insensitive to the chemical identity of the solute for betaine, sorbitol, and triethylene glycol. These solutes appear to modulate binding through their effect on water activity and changes in the hydration of the drug and DNA in the complex reaction, not through a direct interaction with the reactants or the product. The dependence of binding constant on solute concentration can be interpreted as an additional binding of some 50-60 extra solute excluding water molecules by the complex. A water sensitivity of drug binding is further seen from the dependence of binding constants on the type of anion in solution. Anions in the Hofmeister series strongly affect bulk water free energies and entropies. The differences in netropsin analogue binding to DNA with Cl-, F-, and ClO4- are consistent with the effect observed with neutral solutes. The ability to measure changes in water binding associated with a specific DNA interaction is a first step toward correlating changes in hydration with the strength and specificity of binding.
Asunto(s)
ADN/metabolismo , Netropsina/análogos & derivados , Sitios de Unión , Dicroismo Circular , ADN/química , Técnicas In Vitro , Netropsina/química , Netropsina/metabolismo , Concentración Osmolar , Unión Proteica , Soluciones , Termodinámica , AguaRESUMEN
It has become almost axiomatic that protein folding and assembly are dominated by the hydrophobic effect. The contributions from this, and other, hydrophilic interactions can now be better distinguished by direct measurement of forces between proteins. Here we report the measurement of forces between triple helices of type I collagen at different temperatures, pH and solute concentrations. We separate repulsive and attractive components of the net force and analyze the origin of the attraction responsible for the collagen self-assembly. In this case the role of the hydrophobic effect appears to be negligible. Instead, water-mediated hydrogen bonding between polar residues is the most consistent explanation.
Asunto(s)
Colágeno/metabolismo , Animales , Colágeno/química , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Sustancias Macromoleculares , Conformación Proteica , Pliegue de Proteína , Soluciones , Temperatura , Termodinámica , Agua/químicaRESUMEN
Water release coupled to the association of gal repressor with DNA is measured from the sensitivity of the binding constant to the solution osmotic pressure, using neutral solutes that are typically excluded from polar protein and DNA surfaces. Differences in water release for binding of repressor to different sequences are linked with differences in specificity and binding energies. With sucrose, the specific binding of repressor to operator sequences is accompanied by the release of 130 water molecules. No water release is seen for the weak, non-specific binding of repressor to poly(dI-dC).(dI-dC). A difference in the release of six water molecules is seen even for the binding of gal repressor to two different operator sequences that differ in affinity by only a factor of two.
Asunto(s)
ADN/metabolismo , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Agua/metabolismo , Unión Competitiva , Proteínas de Escherichia coli , Cinética , Operón/genética , Concentración Osmolar , Polidesoxirribonucleótidos/metabolismo , Polietilenglicoles , Sacarosa , TermodinámicaAsunto(s)
Sustancias Macromoleculares , Presión Osmótica , Proteínas/química , Agua/metabolismo , ADN/metabolismo , Glucosa/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Hexoquinasa/química , Hexoquinasa/metabolismo , Canales Iónicos/química , Canales Iónicos/metabolismo , Cinética , Concentración Osmolar , Oxígeno/metabolismo , Unión Proteica , Proteínas/metabolismo , Sacarosa/metabolismo , TermodinámicaRESUMEN
We have measured hemoglobin oxygen uptake vs. the partial pressure of oxygen, with independently controlled activities of chloride and water. This control is effected by combining different concentrations of NaCl and sucrose in the bathing solution to achieve: (i) water activities were varied and Cl- activity was fixed, (ii) both water and Cl- activities were varied with a traditional NaCl titration, or (iii) Cl- activities were varied and water activity was fixed by adding compensating sucrose. Within this analysis, the Cl(-)-regulated loading of four oxygens can be described by the reaction Hb.Cl- + 4 O2 + 65 H2O in equilibrium with Hb.4O2.65H2O + Cl-. The dissociation of a neatly integral chloride, rather than the nonintegral 1.6 chlorides inferred earlier from simple salt titration, demonstrates the need to recognize the potentially large contribution from changes in water activity when titrating weakly binding solutes. The single-chloride result might simplify structural considerations of the action of Cl- in hemoglobin regulation.
Asunto(s)
Cloruros/farmacología , Hemoglobinas/metabolismo , Oxihemoglobinas/química , Regulación Alostérica , Hemoglobinas/química , Hemoglobinas/efectos de los fármacos , Humanos , Cinética , Modelos Teóricos , Oxihemoglobinas/efectos de los fármacos , Oxihemoglobinas/metabolismo , Presión Parcial , Sensibilidad y Especificidad , Cloruro de Sodio/farmacología , Termodinámica , AguaRESUMEN
Directly measured forces between DNA helices in ordered arrays have been reduced to simple force coefficients and mathematical expressions for the interactions between pairs of molecules. The tabulated force parameters and mathematical expressions can be applied to parallel molecules or, by transformation, to skewed molecules of variable separation and mutual angle. This "toolbox" of intermolecular forces is intended for use in modelling molecular interactions, assembly, and conformation. The coefficients characterizing both the exponential hydration and the electrostatic interactions depend strongly on the univalent counterion species in solution, but are only weakly sensitive to anion type and temperature (from 5 to 50 degrees C). Interaction coefficients for the exponentially varying hydration force seen at spacings less than 10 to 15 A between surfaces are extracted directly from pressure versus interaxial distance curves. Electrostatic interactions are only observed at larger spacings and are always coupled with configurational fluctuation forces that result in observed exponential decay lengths that are twice the expected Debye-Huckel length. The extraction of electrostatic force parameters relies on a theoretical expression describing steric forces of molecules "colliding" through soft exponentially varying direct interactions.