Dynamic NMR spectral analysis and protein folding: identification of a highly populated folding intermediate of rat intestinal fatty acid-binding protein by 19F NMR.
The folding of intestinal fatty-acid binding protein has been monitored by 19F NMR after incorporation of 6-fluorotryptophan into the protein. The two resonances resulting from the two tryptophans of this protein showed different dependencies on denaturant concentration. One of the resonances was in slow chemical exchange between two resonance frequencies, native and completely unfolded. The changes for this resonance occurred over a denaturant concentration range identical to that monitored by circular dichroism or fluorescence during unfolding. The other resonance continued to show changes at concentrations of denaturant well above that needed to complete the unfolding transition as monitored by optical techniques. Site directed mutagenesis showed that tryptophan-82 was the residue responsible for the unexpected behavior. We conclude, based on complete line-shape analysis, that there are significant concentrations of one or more intermediates in equilibrium with the native and unfolded forms. The structure of the intermediate(s) is more similar to the completely unfolded form of the protein than to the native structure, since little if any secondary structure is present. Further, these structure(s) persist at high denaturant concentrations and may represent local initiating sites in the folding of this beta-sheet protein.
Ropson
I J
IJ
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
Frieden
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D005461:Fluorine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
D001059:Apoproteins; D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D005461:Fluorine