The rat intestinal fatty acid binding protein (IFABP) primarily comprises two β-sheet structures surrounding a large internal cavity. The urea denatured WT protein folds within seconds after dilution to nondenaturing conditions. Replacing a glycine with valine in the turn between the last two β-strands (Gly121Val) slows the folding process by more than three orders of magnitude. After incorporating 4-19F-phenylalanine into the mutant protein, we were able to directly monitor the behavior of the eight phenylalanine side chains in real time during folding using 19F NMR. Specifically, there is a nonnative-like collapse in regions involving three phenylalanine residues (Phe-62, Phe-68, and Phe-93) within milliseconds. At least two distinct NMR peaks were observed, suggesting conformational fluctuations in this region. Formation of this site is followed by formation of native structure of Phe-2 and Phe-17, then by Phe-47, and finally by the cooperative rearrangement of the intermediate structures to the final native structure. It is proposed that the Gly121Val mutation slows the formation of a normal nucleating site, not only slowing overall folding, but also allowing intermediates in regions distant from the mutation to be experimentally observed. Because intermediates involved in protein folding are normally difficult to observe due to their marginal stability, the experimental approach used here may serve as a general method for determining the nature of both early and late steps in protein folding.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Jul 17 2007|
- Folding intermediates
- Nucleation sites
- Turn propensity