Folding and unfolding of a non-fluorescent mutant of green fluorescent protein

Green fluorescent protein ( GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and it remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i. e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/ G67A- GFP, which is unable to efficiently form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of guanidinium hydrochloride ( GdnHCl). The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of 1,1'- bis( 4- anilino- 5- naphthalenesulphonic acid) ( bis- ANS). Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements that S65T/ G67A- GFP folding is largely reversible. During equilibrium folding, the first step is the formation of a molten globule, prone to aggregation.