C-13-NMR OFF-RESONANCE ROTATING-FRAME SPIN-LATTICE RELAXATION STUDIESOF BOVINE LENS GAMMA-CRYSTALLIN SELF-ASSOCIATION - EFFECT OF MACROMOLECULAR CROWDING

The NMR technique of C-13 off-resonance rotating frame spin-lattice re laxation, which provides an accurate assessment of the effective rotat ional correlation time (tau(0,eff)) for macromolecular rotational diff usion, was applied to the study of gamma-crystallin association as a f unction of protein concentration and temperature. Values of the effect ive rotational correlation time for gamma-crystallin rotational diffus ion were obtained at moderate to high protein concentrations (80-350 m g/ml) and at temperatures above, and below, the cold cataract phase tr ansition temperature. With increasing concentration gamma-crystallin w as observed to increasingly associate as reflected by larger values of tau(0,eff) Decreasing temperature in the range of 35 to 22 degrees C was found to result in no change in the temperature corrected value of tau(0,eff) at a gamma-crystallin concentration of 80 mg/ml, whereas a t temperatures of 18 degrees C or below, this parameter was approx. tw ofold larger, suggesting the occurrence of a well defined phase transi tion, which correlated well with the cold cataract phase transition te mperature. At higher protein concentrations, by contrast, tau(0,eff) ( temperature corrected) was found to increase by approx. 1.6- to 2-time s in the temperature interval 35 degrees C to 22 degrees C, a result c onsistent with the dependence of the cold cataract phase transition te mperature on gamma-crystallin concentration. Analysis of intensity rat io dispersion curves, using an assumed model of isodesmic association, permitted the estimation of the association constant characterizing t he aggregation under particular conditions of concentration and temper ature. The significant increase in the value of the association consta nt with moderate increases in protein concentration was rationalized b y invoking the effect of 'macromolecular crowding'. The results obtain ed in this study suggest that in the intact lens, where high protein c oncentrations prevail, gamma-crystallin is unlikely to be found in the monomeric state, but more likely, as a significantly aggregated speci es, representing a broad molecular weight distribution.