Fluorescence spectral changes during the folding of intestinal fatty acid binding protein.
Although large changes in fluorescence intensity are observed during the folding and unfolding of many proteins, it has been difficult to associate these changes with specific structures or with the environmental changes which a particular tryptophan may undergo during these processes. The fluorescence spectral changes that occur during the folding and unfolding of rat intestinal fatty acid binding protein (IFABP) are described here. The intermediate observed during unfolding had spectral characteristics similar to those of unfolded protein, but with somewhat higher intensity. Stopped-flow circular dichroism measurements during unfolding showed that little if any secondary structure was associated with this intermediate. During refolding, the initial fluorescence spectrum was not that of native or unfolded IFABP, suggesting that some structure with intermediate fluorescent properties had formed during the deadtime of mixing. The shape and intensity of this initial spectrum were dependent on the final urea concentration, becoming more native-like at lower final concentrations of denaturant. A simple model for refolding suggests that a portion of the protein molecules obtain native structure and fluorescent characteristics during the deadtime of mixing, and that the remaining protein molecules have spectral characteristics similar to those of the intermediate observed during unfolding. Lower final concentrations of denaturant cause a larger proportion of molecules to follow the rapid refolding pathway. Knowledge of the fluorescence spectral characteristics of the intermediates formed during the folding and unfolding of any protein will improve our understanding of the nature of these structures.
Ropson
I J
IJ
Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Dalessio
P M
PM
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D007422:Intestines / Q000201:enzymology; D008958:Models, Molecular; D018996:Myelin P2 Protein / Q000737:chemistry*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins