Urea induced unfolding followed by tryptophan fluorescence and CD.

Unfolding/refolding transitions were recorded starting with initially folded (0.6 M urea, filled symbols) and unfolded (6.0 M urea, open symbols) CMPK. Tryptophan fluorescence was recorded between 305 and 500 nm. (a) displays the fluorescence intensities between 310 and 319 nm (•) as well as between 350 and 359 nm (▪). Urea dependence of CD at 222 nm is displayed in (b). Raw data of CD and fluorescence intensity was globally fitted to a two state transition, according to Santoro and Bolen [51]. The fits are displayed as solid (initially folded CMPK) and dashed (initially unfolded CMPK) lines (see text). (c) Equilibrium unfolding of *88 mutants. Tryptophan fluorescence (•, intensity at 370 nm) and AEDANS fluorescence(▪, intensity at 470 nm) with excitation at 296 nm. Open symbols indicate single labeled mutants (○, (D+A−); □, (D−A+)), the double labeled mutant (D+A+) is depicted by filled symbols (•▪).