Fluorescence recovery of EGFP and (4<i>S</i>)-FPro-EGFP.

<p>The proteins were denatured by boiling (95°C, 5 min) in 8 M urea and refolded by 100-fold dilution into the buffer without urea (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001680#s3" target="_blank">Methods</a> section for details). Fluorescence emission profiles of (A) (4<i>S</i>)-FPro-EGFP and (B) EGFP upon excitation of the chromophore at 488 nm before denaturation and after 24 h refolding at room temperature. (4<i>S</i>)-FPro-EGFP recovers more than 95% of its fluorescence before denaturation, whereas EGFP recovers only up to 60% of its initial fluorescence (this is in agreement with literature data). (C) The refolding kinetics of both proteins starts with an initial fast phase that is followed by a slow refolding phase. (4<i>S</i>)-FPro-EGFP refolds approximately 2 times faster than EGFP. The percentage of refolding was calculated on the basis of the final constant amount of fluorescence, corresponding to 100% of refolding. Normalized fluorescence in arbitrary units (au) was plotted against time.