Measured change in protein solvation with substrate binding and turnover

Rand, R. P.; Fuller, Nola; Butko, Peter; Francis, G L; Nicholls, Peter

doi:10.1021/bi00074a001

Cited by 132 publications

(127 citation statements)

References 11 publications

(19 reference statements)

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“…Starting a decade ago, a number of papers in which the same approach was used refer to it by the term ''osmotic stress'' (35)(36)(37)(38)(39)(40). The stated aim of these later studies was the determination of the number of stoichiometric water molecules, n, involved in the reaction (35)…”

Section: Modulation Of Protein Reactions By Cosolventsmentioning

confidence: 99%

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Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Timasheff

2002

Proc. Natl. Acad. Sci. U.S.A.

616

663

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Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential exclusion (preferential hydration). The driving force is the perturbation by the protein of the chemical potential of the cosolvent. It is shown that the measured change of the amount of water in contact with protein during the course of the reaction modulated by an osmolyte is a change in preferential hydration that is strictly a measure of the cosolvent chemical potential perturbation by the protein in the ternary water-protein-cosolvent system. It is not equal to the change in water of hydration, because water of hydration is a reflection strictly of protein-water forces in a binary system. There is no direct relation between water of preferential hydration and water of hydration. For the better part of a century, it has been common practice to modulate biochemical (biological) reactions by the addition to the aqueous solvent (dilute buffer) of compounds that were required at high concentration (ϳ0.5 M or higher) to exert their effect. For example, sucrose and glycerol were used to stabilize biological systems, whereas urea and guanidine hydrochloride were used to solubilize coagulated systems and to unfold (denature) proteins. The aim in the use of these additives, referred to as cosolvents, was to displace to the right or left the chemical equilibrium, Reactant º Product. Although this practice was widespread, a theoretical underpinning was lacking until the discovery by Wyman in 1948 of the phenomenon of linked functions (1) and his development of the linkage relationship equations (2, 3), which showed the necessary thermodynamic interdependence between the displacement of a chemical equilibrium and the change in binding of a ligand to the system during the course of the reaction. Complete understanding of the action of cosolvents in such modulation required the combination of the Wyman linkage relations with the multicomponent solution thermodynamics theory developed by Kirkwood and Goldberg (4), Stockmayer (5), and Scatchard (6).The basic Wyman linkage equation states that, at any ligand concentration, m L , the gradient of the equilibrium constant with respect to ligand activity is equal to the change in the binding of the ligand to the biological system during the course of the reaction (at constant temperature and pressure that will be maintained throughout):where K is the equilibrium constant of the reaction, a L is the activity of the ligand (a L ϭ m L ␥ L ), L Prod and L React are the bindings of the ligand to the two end states of the reaction, and m L and ␥ L are the molal concentration and activity coefficient of the ligand. [In our notation, the subscripts W, L, and P refer to water, ligand (cosolvent), and protein (macromolecule), respectively.] Tanford, in 1969 (7), showed that ...

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Section: Modulation Of Protein Reactions By Cosolventsmentioning

confidence: 99%

“…In these studies, the equilibrium was perturbed by addition of a cosolvent, which was stipulated as being an inert excluded osmolyte (35)(36)(37)(38). The equilibrium constant measured as a function of cosolvent concentration was plotted in the form of the Wyman relation (Eq.…”

Section: Modulation Of Protein Reactions By Cosolventsmentioning

confidence: 99%

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Timasheff

2002

Proc. Natl. Acad. Sci. U.S.A.

616

663

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“…For V H 2 O , a value of 18 ml/mol was used. To quantitatively characterize the osmotic pressure dependence of the association process between P450cam and Pdx, we estimated the volume change associated with the osmotically available water in the two states of the equilibrium, ⌬V W , which is defined by Equation 6 (25).…”

Section: Analysis Of the Associationmentioning

confidence: 99%

The Role of Water Molecules in the Association of Cytochrome P450cam with Putidaredoxin

Furukawa

Morishima

2001

Journal of Biological Chemistry

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We have investigated the osmotic pressure dependence of the association between ferric cytochrome P450cam and putidaredoxin (Pdx) to gain an insight into the role of water molecules in the P450cam-reduced Pdx complexation amenable to physiological electron transfer. The association constant was evaluated from the electron transfer rates from reduced Pdx to P450cam. The natural logarithm of the association constant K a was linearly reduced by the osmotic pressure, and osmotic stress yields uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the association in the nonphysiological redox partners P450cam and oxidized Pdx. Although general proteinprotein associations proceed through dehydration around the complex interface, the interfacial waters could mediate hydrogen-bonding interactions. Therefore, about 10 more interfacial waters imply an additional water-mediated hydrogen-bonding network in the P450cam⅐reduced Pdx complex, which does not exist in the complex with oxidized Pdx. It is also possible that the water-mediated hydrogen-bonding interactions support a high P450cam affinity for reduced (K a ‫؍‬ 0.83 M ؊1 ) relative to oxidized (K a ‫؍‬ 0.058 M ؊1 ) Pdx. This study points to a novel role of solvents in assisting redox state-dependent interaction between P450cam and Pdx.

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“…The result is that by reducing the activity of water, the presence of an osmotically active osmolyte inhibits protein conformational changes in which there is a net uptake of water and favors processes in which a protein releases water to the bulk aqueous medium. Osmolytes, which are so small that they are not excluded from any clefts or pockets on the protein surface, will not be osmotically active (12). Recently, we employed this technique to show that the MI to MII transition of photoactivated rhodopsin is dependent upon water activity, with the MII conformation binding 35 Ϯ 2 fewer water molecules than MI (13).…”

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confidence: 99%

Effect of Ethanol and Osmotic Stress on Receptor Conformation

Mitchell

Litman

2000

Journal of Biological Chemistry

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The combined effects of ethanol and osmolytes on both the extent of formation of metarhodopsin II (MII), which binds and activates transducin, and on acyl chain packing were examined in rod outer segment disc membranes. The ethanol-induced increase in MII formation was amplified by the addition of neutral osmolytes. This enhancement was linear with osmolality. At 360 milliosmolal, the osmolality of human plasma, 50 mM ethanol was 2.7 times more potent than at 0 osmolality, demonstrating the importance of water activity in in vitro experiments dealing with ethanol potency. Ethanol disordered acyl chain packing, and increasing osmolality enhanced this acyl chain disordering. Prior osmotic stress data showed a release of 35 ؎ 2 water molecules upon MII formation. Ethanol increases this number to 49 water molecules, suggesting that ethanol replaces 15 additional water molecules upon MII formation. Amplification of ethanol effects on MII formation and acyl chain packing by osmolytes suggests that ethanol increases the equilibrium concentration of MII both by disordering acyl chain packing and by disrupting rhodopsin-water hydrogen bonds, demonstrating a direct effect of ethanol on rhodopsin. At physiologically relevant levels of osmolality and ethanol, about 90% of ethanol's effect is due to disordered acyl chain packing.One of the proposed modes of action of ethanol on biological macromolecules and membranes involves the replacement of hydrogen-bonded water by ethanol (1, 2). Based on the hydrogen bonding capability of both water and ethanol, it is postulated that they compete for hydrogen bonding sites on the surface of lipids and proteins. Thus, this action of ethanol is equally applicable to mechanisms of ethanol action involving both lipid-mediated and direct protein interactions. The general importance of changes in hydration in enzyme activity is widely acknowledged (3, 4), but there is little detailed knowledge regarding how replacement of water by ethanol in specific protein-water hydrogen bonds might alter protein conformational equilibria or function. In addition, the physical properties of the surrounding phospholipid bilayer are known to modulate membrane function. Ethanol-induced changes in phospholipid hydrogen bonding have been observed to change acyl chain packing in pure lipid bilayers (5-7). Thus, the function of integral membrane receptors could be especially susceptible to the disruption of hydrogen-bonded water by ethanol.Thorough examination of the interplay between ethanol and hydration in modulating membrane receptor function requires the ability to separately analyze the effects of ethanol on both protein structure and bilayer physical properties and to be able to correlate the observed changes. Previously, we examined the effects of ethanol (8) and a series of n-alkanols (9) on both the MI 1 -MII conformational equilibrium and acyl chain packing in the surrounding bilayer. In the present work, the effects of ethanol on the MI-MII equilibrium and acyl chain packing are reexamined as a functi...

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Measured change in protein solvation with substrate binding and turnover

Cited by 132 publications

References 11 publications

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

The Role of Water Molecules in the Association of Cytochrome P450cam with Putidaredoxin

Effect of Ethanol and Osmotic Stress on Receptor Conformation

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