Figure 4. Hemolytic activities and biophysical properties of mutant pneumolysins.

(A) hemolysis of sheep erythrocytes by selected pneumolysin mutants. (B) Fluorescence spectra of folded and unfolded pneumolysin (in 2M guanidine-HCl) and pneumolysin with liposomes. The fluorescence spectra of all mutants were comparable to that of the wild-type and exhibited similar changes with respect to the fluorescence intensity and λ_{em, max}. (C,D), denaturation of wild-type and mutant pneumolysins measured by changes in fluorescence intensity (C) and λ_{em, max} (D). The unfolding transition measured by the change in λ_{em, max} corresponded approximately to the decrease in fluorescence observed in (C). (E) cholesterol-mediated inhibition of haemolysis by wild-type and mutant pneumolysins. Data are average of six measurements.