title: 13C-NMR off-resonance rotating frame spin-lattice relaxation studies of bovine lens gamma-crystallin self association: effect of 'macromolecular crowding'.
abstract: The NMR technique of 13C off-resonance rotating frame spin-lattice relaxation, which provides an accurate assessment of the effective rotational correlation time (tau 0, eff) for macromolecular rotational diffusion, was applied to the study of gamma-crystallin association as a function of protein concentration and temperature. Values of the effective rotational correlation time for gamma-crystallin rotational diffusion were obtained at moderate to high protein concentrations (80-350 mg/ml) and at temperatures above, and below, the cold cataract phase transition temperature. With increasing concentration gamma-crystallin was 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 at temperatures of 18 degrees C or below, this parameter was approx. twofold larger, suggesting the occurrence of a well defined phase transition, which correlated well with the cold cataract phase transition temperature. At higher protein concentrations, by contrast, tau 0, eff (temperature corrected) was found to increase by approx. 1.6- to 2-times in the temperature interval 35 degrees C to 22 degrees C, a result consistent with the dependence of the cold cataract phase transition temperature on gamma-crystallin concentration. Analysis of intensity ratio dispersion curves, using an assumed model of isodesmic association, permitted the estimation of the association constant characterizing the aggregation under particular conditions of concentration and temperature. The significant increase in the value of the association constant with moderate increases in protein concentration was rationalized by invoking the effect of 'macromolecular crowding'. The results obtained in this study suggest that in the intact lens, where high protein concentrations prevail, gamma-crystallin is unlikely to be found in the monomeric state, but more likely, as a significantly aggregated species, representing a broad molecular weight distribution.
authors: Department of Chemistry and Biochemistry, Sinsheimer Laboratories, University of California, Santa Cruz 95064.
authors: A
authors: A
authors: Stevens
authors: S X
authors: SX
authors: Wang
authors: G H
authors: GH
authors: Caines
authors: T
authors: T
authors: Schleich
mesh_terms: D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003459:Crystallins / Q000737:chemistry*; D004058:Diffusion; D007908:Lens, Crystalline / Q000737:chemistry*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D013696:Temperature
chemical_list: D003459:Crystallins; D046911:Macromolecular Substances