Osmotic stress measurements

More recently, osmotic stress measurements have been used to determine differences in the number of sequestered waters among the complexes of X cro repressor with one cognate and four noncognate sequences (47). It has been found that the number of sequestered water molecules for a particular cro-DNA complex correlates linearly with the binding free energy, AG, with each extra bound water, a loss of 150 cal mol in AG. 

Osmotic stress measures forces corresponding to pressures over six decades, from 0.01 to 10,000 atm. With X-ray diffraction by ordered arrays, as shown in Figure 1, molecular dimensions and intermolecular spacings can be obtained with accuracies often better than 0.2 A reproducibility. Alternatively, with probes of ionic channel conductance or of protein activity, the behavior of single molecules can be observed and the osmotically sensitive part of the underlying structural transformation can be extracted. Consequently, the change in the volume of associated water as the system goes between active and inactive forms can also be extracted. 

We now describe some common features of swelling at different levels from osmotic stress measurements on several kinds of experimental systems. 

Parsegian, V. A., Rand, R. P. and Fuller, N. L., Direct osmotic stress measurements of hydration and electrostatic double-layer forces between bilayers of double-chained ammonium acetate surfactants, J. Phys. Chem., 95, 4777-4782 (1991). 

Measures, J. C. Reactions of Microbial Cells to Osmotic Stress, in 14. ... 

Complementary to the SFA experiments, SFM techniques enabled direct, non-destructive and non-contact measurement of forces which can be as small as 1 pN. Compared to other probes such as optical tweezers, surface force balance and osmotic stress [378-380], the scanning force microscope has an advantage due to its ability in local force measurements on heterogeneous and rough surfaces with excellent spatial resolution [381]. Thus, a force-distance dependence measured from a small surface area provides a microscopic basis for understanding the macroscopic interfacial properties. Furthermore, lateral mapping... 

The influence of various cosolvents on protein stability has been discussed by Timasheff [50]. There has been a considerable debate in the literature on the number of water molecules that are taking part in protein-protein or protein-DNA interactions as estimated by various methods. A recent theoretical analysis suggests that the osmotic stress method may overestimate the number of waters involved [51]. These models assume that the cavities that are formed at the interface between macromolecules do not contribute to the measured volume changes as suggested by Silva and Weber [31]. 

Another aspect of current interest associated with the lipid-water system is the hydration force problem.i -20 When certain lipid bilayers are brought closer than 20-30 A in water or other dipolar solvents, they experience large repulsive forces. This force is called solvation pressure and when the solvent is water, it is called hydration pressure. Experimentally, hydration forces are measured in an osmotic stress (OS) apparatus or surface force apparatus (SFA)2o at different hydration levels. In OS, the water in a multilamellar system is brought to thermodynamic equilibrium with water in a polymer solution of known osmotic pressure. The chemical potential of water in the polymer solution with which the water in the interlamellar water is equilibrated gives the net repulsive pressure between the bilayers. In the SEA, one measures the force between two crossed cylinders of mica coated with lipid bilayers and immersed in solvent. 

Experimental Results. The DLVO theory, which is based on a continuum description of matter, explains the nature of the forces acting between membrane surfaces that are separated by distances beyond 10 molecular solvent diameters. When the interface distance is below 10 solvent diameters the continuum picture breaks down and the molecular nature of the matter should be taken into account. Indeed the experiment shows that for these distances the forces acting between the molecularly smooth surfaces (e.g., mica) have an oscillatory character (8). The oscillations of the force are correlated to the size of the solvent, and obviously reflect the molecular nature of the solvent. In the case of the rough surfaces, or more specifically biomembrane surfaces, the solvation force displays a mono tonic behavior. It is the nature of this solvation force (if the solvent is water, then the force is called hydration force) that still remains a puzzle. The hydration (solvation) forces have been measured by using the surface force apparatus (9) and by the osmotic stress method (10, II). Forces between phosphatidylcholine (PC) bilayers have been measured using both methods and good agreement was found. 

For either the two- or the three-dimensional geometry of molecular arrays, the free energy of assembly, AG, is measured as the integral of osmotic stress versus solvent volume V ... 

Figure 1. Schematic of the general method of using osmotic stress to measure the contribution of water to the energetics of a molecular array or of a...

Figure 2. Illustration of how the osmotic stress method is used to measure the volume of water associated with the opening and closing of a membrane...

Figure 4. The osmotic stress method used to measure net repulsive pressure between membranes as it varies with membrane separation. Solid symbols represent stearoyloleoylphosphatidylcholine open symbols represent palmitoyl-oleoylphosphatidylethanolamine.

Under osmotic stress, the aqueous compartment shrinks and the mono-layer around the water cylinder bends. The osmotic work can be translated into monolayer- and bilayer-bending moduli (7) to yield a bilayer value of about 1.5 X 10 12 erg, which is in qualitative agreement with measurements from bilayer mechanics (38). It is possible to refine the analysis of Hn dehydration to include the change in area per lipid molecule and to extract both a bending modulus and a lateral compressibility moduli. This refinement gives moduli in close quantitative agreement with other measures of mechanical deformation (39). 

Figure 6. Osmotic stress used to measure the intermacromolecular force as it varies with molecular distance.

H. Coll and F. H. Stress, Determination of molecular weights by equilibrium osmotic pressure measurements, in Characterization of Macromolecular Structure, Natl. Acad. Sci. U.S. Publ. 1573, Washington, D.C., 1968. 

In parallel, another important (although less direct) technique for measuring forces between macromolecules or lipid bilayers was developed, namely, the osmotic stress method [39-41]. A dispersion of vesicles or macromolecules is equilibrated with a reservoir solution containing water and other small solutes, which can freely exchange with the dispersion phase. The reservoir also contains a polymer that cannot diffuse into the dispersion. The polymer concentration determines the osmotic stress acting on the dispersion. The spacing between the macromolecules or vesicles is measured by X-ray diffraction (XRD). In this way, one obtains pressure-versus-distance curves. The osmotic stress method is used to measure interactions between lipid bilayers, DNA, polysaccharides, proteins, and other macromolecules [36]. It was particularly successful in studying the hydration... 

The interactions between biopolymers are experimentally measured byusing a range of techniques. Osmotic stress and osmotic pressme tedmiques involve the determination of intermolecirlar interactions between biopolymers that are crowded within ordered phases (e.g., liquid crystals, colloidal arrays, lamdlae, or stacked bilayers) (The crowding in such phases is typically modrrlated by changing the water adivity within the ordered phase by contaaing the biopolymer... 

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