Figure 6. Putative scheme representing GFP unfolding by PAN.

(A) At the beginning of the reaction PAN is in a relaxed, extended state and all GFP molecules are in their native state with maximum fluorescence. (B) After a few minutes PAN reaches its maximum activity and destabilizes/unfolds GFP molecules. GFP molecules are either released in a destabilized, yet fluorescent state or in a completely denatured, unfluorescent state and form first, small aggregates. (C) At a later state all natively folded GFP molecules have disappeared and all destabilized or denatured GFP molecules are clustered within aggregates. Occasionally, PAN interacts with clusters and denatures the destabilized members.