CHARACTERIZATION OF THE EARLY INTERACTIONS ON THE FOLDING PATHWAY OF THE ILEAL LIPID-BINDING PROTEIN BY 19F-NMR
Open Access
- Author:
- Basehore, Heather Kenney
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 09, 2007
- Committee Members:
- Ira Joseph Ropson, Committee Chair/Co-Chair
Dr Michael Pishko, Committee Member
Cara Lynne Schengrund, Committee Member
Michael Verderame, Committee Member - Keywords:
- b-sheet proteins
19F-NMR
protein folding - Abstract:
- Abstract: Determining whether two structurally related proteins fold via similar mechanisms is an important question in structural biology. Intracellular lipid-binding proteins (iLBP’s) are a large family of proteins that share a common ?-barrel structure in spite of very low sequence similarities. One member of this family, intestinal fatty acid-binding protein (IFABP) has been shown to fold by a mechanism of specific hydrophobic collapse in a core structural region. These kinetic folding intermediates differ from intermediates observed for the structurally related protein, ileal lipid-binding protein (ILBP). There is some evidence that both proteins initiate folding by the same mechanism, but that stable folding intermediates do not accumulate to the same level for both. The proposed folding mechanism of IFABP is supported by equilibrium unfolding studies by 19F-NMR. Residues in contact with each other in the native protein retain elements of structure at denaturant concentrations much higher than the unfolding midpoint concentrations. This is manifested in the 19F-NMR spectra in the form of NMR spectral changes that differ in response to denaturant depending on the residue’s role in the folding pathway. Similar analysis of ILBP demonstrates that some residues in this protein also display non-uniform behavior by 19F-NMR with increasing denaturant, although the residues responsible for this behavior were not identified in the initial analysis. By labeling the protein with 19F one residue at a time, assignment of the peak resonances for each 19F-Phe in ILBP have now been made. The results of these 19F-NMR experiments reveal that some of the interactions that occur early in folding may be conserved, but the specific nature of the initiating reactions differs for these two related proteins.