Abstract

The structure of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) exhibits a beta-clam topology comprised of two five-stranded antiparallel beta-sheets surrounding a large solvent-filled cavity into which the ligand binds. It also contains two alpha-helices that span residues E15-A32 and join beta-strands A and B. This helical domain is conserved in all proteins of this family for which structures have been determined. In order to assess the structural and functional role of the helical domain, we engineered a variant of I-FABP by deleting residues 15-31 and inserting a Ser-Gly linker after residue 14. Circular dichroism measurements indicated that this I-FABP variant, termed delta 17-SG, has a high beta-sheet content similar to that of the wild-type protein. Two-dimensional NMR spectra of delta 17-SG revealed patterns similar to those observed for wild-type I-FABP, except for the selective absence of resonances and through-space interactions assigned to the helical domain. The delta 17-SG variant was less stable to denaturant than wild-type I-FABP, but the folding-unfolding transition was highly cooperative and reversible. Taking into account the lower stability, the refolding kinetics of delta 17-SG were essentially identical to those of wild-type. We conclude that delta 17-SG is a helix-less, essentially all-beta-sheet variant of I-FABP and that the helical domain is not a required element of the beta-clam topology of I-FABP. In addition, the helical domain does not appear to serve as a nucleation site for the refolding process. As shown in the accompanying paper [Cistola, D. P., Kim, K., Rogl, H., & Frieden, C. (1996) Biochemistry 35, 7559-7565], the helices may function to regulate the kinetics and energetics of ligand binding. Study holds ProTherm entries: 5036 Extra Details: additive : EDTA(0.25 mM), antiparallel beta-sheets; solvent-filled cavity; functional role,helical domain; folding-unfolding transition; cooperative

Submission Details

ID: rZygnFM23

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

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