Quantitative NMR study of heat-induced aggregation of eye-lens crystallin proteins under crowding conditions

The eye lens contains a highly concentrated, polydisperse mixture of crystallins, and a loss in transparency during cataract formation is attributed to the aggregation of these proteins. Most biochemical and biophysical studies of crystallins have been performed in diluted samples because of various physical limitations of the respective method at physiological concentrations of up to 200–400 mg/ml. We introduce a straightforward proton NMR transverse relaxometry method to quantify simultaneously proteins in the dissolved and aggregated states at these elevated concentrations, because these states significantly differ in their transverse relaxation properties. The key feature of this method is a direct observation of the protein signal in a wide range of relaxation delays, from few microseconds up to few hundred milliseconds. We applied this method to follow heat-induced aggregation of bovine α- and γB-crystallin between 60 and 200 mg/ml. We find that at 60 °C, a temperature where both crystallins still comprise a native tertiary structure, γB-crystallin aggregated at these high protein concentrations with a time constant of about 30–40 h. α-crystallin remained soluble at 60 mg/ml but formed a transparent gel at 200 mg/ml. This quantitative NMR method can be applied to investigations of other proteins and their mixtures under various aggregation conditions.