To initiate aggregation, the labeled P39A tetra-Cys CRABP is destabilized by the addition of 10 mM HEPES containing 8.5 M urea (pH 7.8) pre-incubated at 37° to a final urea concentration of 1.8 M. Samples, usually 200 to 250 ßl total volume, are incubated without stirring and subjected periodically to fluorescence measurement at 530 nm upon excitation at 500 nm (bandwidth 2 nm). Prior to withdrawal of an aliquot for measurement (120 ßl), the reaction mixture is vortexed gently to ensure a homogeneous distribution of the aggregates over the entire volume of the sample. The temperature of the cuvette holder is maintained at 37° with a water bath. Blank samples treated in the same way but lacking protein serve as negative controls, and their values are subtracted from that of the sample. At different time points after initiation of the in vitro aggregation of P39A tetra-Cys CRABP, osmolytes (proline, glycine betaine, or trehalose) are added to a final concentration of 500 mM, and the aggregation is monitored by FlAsH fluorescence. The rationale for using a 500 mM concentration of the osmo-lytes in the in vitro experiments is based on literature data showing that osmotic stress induced by the osmolality ofthe nutrient medium used in our in vivo experiments causes an accumulation of approximately 500 mM osmolyte (Cayley and Record, 2003).
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