Intestinal fatty acid binding protein: the folding mechanism as determined by NMR studies.
The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.
Hodsdon
M E
ME
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Frieden
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C
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D007413:Intestinal Mucosa / Q000737:chemistry / Q000378:metabolism; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
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; D006859:Hydrogen; D014508:Urea; D003903:Deuterium