The intestinal fatty acid binding protein: The role of turns in fast and slow folding processes

Krishnananda Chattopadhyay, Shi Zhong, Syun Ru Yeh, Denis L. Rousseau, Carl Frieden

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31 Scopus citations


The intestinal fatty acid binding protein is one of a family of proteins that are composed of two β-sheets surrounding a large interior cavity into which the ligand binds. Glycine residues occur in many of the turns between adjacent antiparallel β-strands. In previous work, the effect of replacing these glycine residues with valine has been examined with stopped flow instrumentation using intrinsic tryptophan fluorescence spectroscopy [Kim and Frieden (1998) Protein Sci. 7, 1821-1828]. To resolve the burst phase missing in the stopped flow measurements, these valine mutants have been reexamined with sub-millisecond continuous flow instrumentation. Some of the glycine residues have also been replaced with proline, and the folding reactions of these proline mutants have been compared with those of their valine counterparts. In all cases, the stability of the protein is decreased, but some turns appear to be more critical for final structure stabilization than others. Surprisingly, the rate constants observed for all the mutants measured by sub-millisecond continuous flow methods are quite similar (1400-3000 s-1), and in all the mutants, there is a shift in the fluorescence emission maximum from that of the unfolded protein to lower wavelengths, suggesting some collapse of the unfolded state within 200 μs. In contrast to the rate constants observed for the initial folding events measured by the sub-millisecond continuous flow method, the rate constants for the slower phase observed in the stopped flow instrument vary widely for the different mutants. The latter step appears to be related to side chain stabilization rather than secondary structure formation. It is also shown that the ligand binds tightly only to the native protein and not to any intermediate forms.

Original languageEnglish
Pages (from-to)4040-4047
Number of pages8
Issue number12
StatePublished - Mar 26 2002


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