Fluorescence Spectral Changes During the Folding of Intestinal Fatty Acid Binding Protein

Abstract

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.

Structural Events during the Refolding of an All β-Sheet Protein

“…1-Anilino-8-naphthalenesulfonic acid-NH 4 ϩ salt was purchased from Sigma. Ultrapure urea was procured from Merck, and heparin-Sepharose was from Amersham Pharmacia Biotech.…”

“…Ultrapure urea was procured from Merck, and heparin-Sepharose was from Amersham Pharmacia Biotech. Labeled 15 NH 4 Cl and urea-d 4 were obtained from Cambridge Isotope Laboratories. All other chemicals used were of high quality analytical grade.…”

Structure, Stability, and Aggregation of Paired Helical Filaments from Tau Protein and FTDP-17 Mutants Probed by Tryptophan Scanning Mutagenesis

“…The positions of the excitation and emission maxima, 288 and 354 nm, indicate that the Trp residue is almost fully exposed to the solvent in the soluble state and not buried in the interior of the protein. The blue shift following PHF aggregation is explained by an increased hydrophobicity in the local environment (30,31); it suggests that Trp 310 becomes buried in a hydrophobic pocket within the PHFs.…”

Folding mechanism of three structurally similar β-sheet proteins

“…15 Working urea solutions (9 M) were made from the 10-M stock on the day of the experiment by adding 25 mM NaPO 4 , 75 mM NaCl, 0.1 mM EDTA at pH 8.0. The urea concentration was determined by refractive index measurements using a Milton Roy Abbe-3C refractometer at 25°C in conjunction with an equation-relating refractive index to concentration.…”

“…A Jasco J-710 spectropolarimeter in conjunction with a RX1000 stopped-flow apparatus (Applied Photophysics) was used to monitor intensity changes at 218 nm (2 nm bandpass). 15 Five parts of denaturant solution were mixed with one part protein solution. Final protein concentrations were 32-33 µM.…”