Figure 21.3 Glycine betaine is not able to completely abolish the aggregation of P39A tetra-Cys CRABP in vivo and in vitro. (A) Time evolution of the bulk FlAsH fluorescence at 530 nm (excitation 500 nm) of E. coli BL21(DE3) cells expressing P39A tetra-Cys CRABP, cultured in a low salinity medium and in high salinity (300 mM) supplied with 20 mM glycine betaine (GB). For comparison, signal from fully soluble tetra-Cys CRABP is also shown. (B) Time evolution of the in vivo aggregation of P39A tetra-Cys CRABP monitored by the bulk FlAsH fluorescence signal of labeled cells, under depleted (-ProP) and upregulated (+ProP) ProP transporter conditions in the E. coli strain WG710. The expression of soluble tetra-Cys CRABP is not influenced by the amount of the glycine betaine influx; it remains the same in the absence (-ProP) and upon accumulation of glycine betaine through the ProP transporter (+ProP). (C) Glycine betaine (500 mM) added at different times during the in vitro aggregation of 15 mM labeled P39A tetra-Cys CRABP. The in vitro P39A tetra-Cys CRABP aggregation was monitored by FlAsH fluorescence (unpublished results of Ignatova and Gierasch).
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