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