Turn scanning by site‐directed mutagenesis: Application to the protein folding problem using the intestinal fatty acid binding protein

Abstract

We have systematically mutated residues located in turns between P-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all P-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel P-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process . Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.Keywords: P-sheet protein folding and stability; fatty acid binding; turn mutationsThe intestinal fatty acid binding protein (IFABP) is a small (15,000 Da) monomeric protein that belongs to a class of proteins that are primarily P-sheet and bind a diverse group of ligands (fatty acids, retinoids, and bile salts) into a large central cavity in the interior of the protein. The structure of the protein has been examined both by X-ray Sacchettini & Gordon, 1993;Banaszak et al., 1994) and NMR (Hodsdon et al., 1996;Hodsdon & Cistola, 1997a, 1997bZhang et al., 1997) methods. It is an excellent model protein for folding studies, not only because of its small size and P-sheet structure, but also because it contains no cysteine or proline residues. In a previous paper , we investigated the role of the turn between two antiparallel Reprint requests to: C. Frieden, Department of Biochemistry and Molecular Biophysics, Box 8231, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63 I 1 0 e-mail: frieden@biochem. wustl.edu.Abbreviutions: IFABP, rat intestinal fatty acid binding protein; Gdn, guanidine hydrochloride; EDTA, ethylenediamine tetraacetic acid; DAUDA, 1 1 -((5-dimethylaminonaphth...

Observation of sequential steps in the folding of intestinal fatty acid binding protein using a slow folding mutant and19F NMR

“…1C), consistent with an earlier study on the unfolding by guanidine-HCl (12). Further, as a function of the urea concentration, the G121V mutant showed much smaller chemical shift changes than the WT before global unfolding, although their chemical shifts change in the same direction (data not shown).…”

“…This analysis is consistent with the theory of protein folding kinetics (6,23,24) in that the major determinant (the contact order) of protein folding rates is conserved for the G121V mutant and the WT because they form the same structure as suggested by 19 F-NMR (Fig. 1B) and near UV-CD studies (12), whereas the stability of transient metastable states was modulated by turn propensity and local hydrophobicity around the residue at position 121. The data also show that intermediates experience multiple conformations.…”

“…Glycine 121 is located in a turn between the last two ␤-strands of IFABP. Replacing this glycine with valine has been shown to dramatically slow the refolding process (12). The present 19 F-NMR study shows a rapid collapse involving at least three phenylalanines, all of which are distant from the mutation site.…”

“…The phenylalanine auxotroph DL39 was used to express 4-19 F-Phe-labeled G121V-IFABP according to established procedures (13,28). The 4-19 F-labeled IFABP mutant was expressed and purified as described elsewhere (12). The labeling efficiency was Ͼ95% as determined by mass spectroscopy (data not shown).…”

Fluorine-19 NMR Studies on the Acid State of the Intestinal Fatty Acid Binding Protein

“…Other residues did not show significant line width changes in the pH range studied. These observations are consistent with previous studies (14,15) showing that two regions appear to be involved in the early stage of urea folding, one is close to D-E turn, the other one is close to I-J turn and helical region ( Figure 1). …”