Investigation of gammaE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering.

alpha-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between alpha-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (gammaE-crystallin) with alpha-crystallin (alpha(H)) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(H)/D(2)O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2)O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.

Structural changes in alpha-crystallin and whole eye lens during heating, observed by low-angle X-ray diffraction.

Whole eye lens and alpha-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 degrees C to 70 degrees C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 degrees C to 45 degrees C but increased at 50 degrees C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of alpha-crystallin gels at near-physiological concentration (approximately 300 mg/ml), so it is reasonable to assume that the alpha-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens alpha-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the alpha-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 degrees C to 45 degrees C, followed by an accelerated increase from 45 degrees C to 70 degrees C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for alpha-crystallin solutions at a variety of lower concentrations.

The ordering of corneal collagen fibrils with increasing ionic strength.

The fixed stromal charge of bovine corneas, osmotically clamped at physiological hydration, was altered by regulating the amount of chloride ions bound to the matrix. We measured the local fibrillar collagen order using X-ray diffraction methods. As the bound anions increased up to physiological values, the local fibrillar order increased to an optimal value. The coherence distance (t) approximately doubles to a maximum value (409 nm) from 10 mM NaCl to 154 mM NaCl. This then slowly decreased as the bathing solution increased to 1000 mM. In contrast the diameter of the collagen fibrils were minimal at physiological NaCl.

Neutron and X-ray scattering by ox corneal stroma differentially loaded with bound anions.

Ox corneas at near physiological hydration were subjected to two variables: the amount of chloride ions bound to them and exposure of various mixtures of H(2)O/D(2)O as solvent. The preparations were then exposed to a neutron beam and the contrast match points, at which the collagen fibrils of the corneal stroma most nearly matched the scattering density of the various H(2)O/D(2)O mixtures, were measured. In both cases of high and low bound chloride, the contrast match points of the collagen fibril were equal, indicating that there were no significant changes in the water of electrostriction at the fibril surface when chloride ions bind to the stroma. The data suggest that the ligands which bind anions to corneal stroma are not located at the collagen fibril surface. When the chloride binding ligands were extracted from the corneal stroma there were significant changes in the structure of the fibrils. We suggest that the chloride binding ligands may be located within the collagen fibril.

The effect of temperature on the Donnan potentials in biological polyelectrolyte gels: cornea and striated muscle.

The effect of temperature on the fixed electric charge in biological polyelectrolyte gels was studied between 10 and 35 degrees C using the Donnan microelectrode technique. Two tissues; cornea and striated muscle were used. In cornea, there is a gentle and uniform decrease in fixed charge over the temperature range. In rigor muscle, there is a dramatic step-function decrease in charge at around 28 degrees C. There is a charge decrease in relaxed muscle at around the same temperature, but the step function is less distinct. The significance of these different experimental relationships is discussed in relation to the Saroff model for ion binding to proteins, linked to the possible disordering effects of excess electric charge. The diverse effects in these systems are important for the physiological functions of the different tissues.