FLUORESCENCE SPECTRAL CHANGES DURING THE FOLDING OF INTESTINAL FATTY-ACID-BINDING PROTEIN

Although large changes in fluorescence intensity are observed during t he folding and unfolding of many proteins, it has been difficult to as sociate these changes with specific structures or with the environment al changes which a particular tryptophan may undergo during these proc esses. The fluorescence spectral changes that occur during the folding and unfolding of rat intestinal fatty acid binding protein (IFABP) ar e described here. The intermediate observed during unfolding had spect ral characteristics similar to those of unfolded protein, but with som ewhat higher intensity. Stopped-flow circular dichroism measurements d uring unfolding showed that little if any secondary structure was asso ciated with this intermediate. During refolding, the initial fluoresce nce spectrum was not that of native or unfolded IFABP, suggesting that some structure with intermediate fluorescent properties had formed du ring the deadtime of mixing. The shape and intensity of this initial s pectrum were dependent on the final urea concentration, becoming more native-like at lower final concentrations of denaturant, A simple mode l for refolding suggests that a portion of the protein molecules obtai n native structure and fluorescent characteristics during the deadtime of mixing, and that the remaining protein molecules have spectral cha racteristics similar to those of the intermediate observed during unfo lding. Lower final concentrations of denaturant cause a larger proport ion of molecules to follow the rapid refolding pathway. Knowledge of t he fluorescence spectral characteristics of the intermediates formed d uring the folding and unfolding of any protein will improve our unders tanding of the nature of these structures.