Steady-state and time-resolved fluorescence studies of the intestinal fatty acid binding protein.
The intestinal fatty acid binding protein contains two tryptophan residues (Trp6 and Trp82) both of which have been shown by X-ray and NMR methods to be buried in hydrophobic clusters. By using a combination of steady-state and time-resolved fluorescence experiments, we have deconvoluted the lifetime weighted contribution of each of the tryptophans to the steady-state fluorescence quantum yield. While Trp82 has been implicated in an intermediate that appears at relatively high denaturant concentrations, the variation of the lifetime weighted contribution of Trp6 with urea or guanidium hydrochloride shows formation of an intermediate state at low concentrations of the denaturant before the actual unfolding starts. Trp82 did not show similar behavior. Fluorescence quenching experiments by acrylamide show that while Trp6 in the native protein is less solvent-exposed, its accessibility is increased significantly at low urea concentration indicating that the early intermediate state is partially unfolded. Time-resolved anisotropy experiments indicate that the volume of the partially unfolded intermediates is larger than the native protein and lead to the speculation that the last step of the protein folding might be the removal of solvent molecules from the protein.
Chattopadhyay
Krishnananda
K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA.
Frieden
Carl
C
D000143:Acids / Q000494:pharmacology; D018360:Crystallography, X-Ray; D050556:Fatty Acid-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D000143:Acids; D050556:Fatty Acid-Binding Proteins; D014364:Tryptophan; D014508:Urea