Refolding of labeled CMPK mutants.

AEDANS attached at different positions to CMPK (see Fig. 1) serves as FRET acceptor with the single tryptophan as FRET donor. This leads to decrease in fluorescence of the (Trp) donor (a) and increase in fluorescence of (AEDANS) the acceptor (b) when the fluorophores approach upon folding. Excitation was performed at 296 nm. Gray: Trp signal of D+A− mutants; blue: *88; green: *197; red: *208. The light-colored traces in (b) correspond to the AEDANS-signal of D-A+ mutants. A double exponential is no longer sufficient to describe the data, and a triple exponential has to be used instead. The opposing data-courses within the first second illustrate the effect in energy transfer (increase in *88, decrease in *208 and no change in *197). The transfer efficiencies for each mutant based on donor quenching and sensitized acceptor-emission calculated from global fit results of single and double labeled mutants is displayed in (c). The major changes are linked to the burst, fast and slow phase, while the intermediate phase shows almost no change in transfer efficiency.