Functional and conformational characterization of new mutants of heart fatty acid-binding protein

Abstract

In this study we investigated the possible involvement of several amino acids (not located in the ligand-binding centre) in fatty acid binding and conformational stability of heart fatty acid-binding protein (H-FABP). We prepared recombinant human H-FABP proteins with mutations in the hydrophobic patch (Phe(4), Trp(8) and Phe(64)), portal region (Phe(16)), hinge region (Leu(66), Gly(67)), second portal region (Glu(72)) and at the protein surface (Lys(21)) respectively. Oleic acid-binding affinity and conformational stability of human H-FABP are significantly decreased or completely lost by mutation of Trp(8) or Phe(16). NMR spectra confirmed that these residues are important for the stability of the protein fold. Substitution of Phe(4) or Phe(64) resulted in less stability, but oleic acid-binding affinity was not affected. Mutation of Lys(21) had no effect on either structural integrity or fatty acid-binding affinity. Replacement of Leu(66) or Gly(67) did not affect fatty acid binding, but protein stability was reduced. Finally, mutation of Glu(72) to Ser caused no change of affinity, but NMR spectra and urea-denaturation curves showed the extremely poor stability of this mutant. In conclusion, no relationship was observed between fatty acid-binding affinity and conformational stability.

A novel fatty acid binding protein produced by teratocytes of the aphid parasitoid Aphidius ervi

“…Ae ‐FABP is, to our knowledge, the first insect FABP identified that has an extracellular localization, as supported by its immunodetection on Western blots of proteins present in the cell‐free plasma of parasitized host aphids. It is of interest to note that the putative signal peptide is retained by the protein found in vivo , likely because this region of p15 contains the amino acid residue Phe16, which seems to be of crucial importance for ligand binding in other FABPs (Prinsen & Veerkamp, 1996; Zimmerman et al ., 1999). This further corroborates the hypothesis that some proteins may need to retain the signal peptide in order to maintain their proper function (Blobel & Dobberstein, 1975).…”

“…Detailed structure‐activity studies indicate that mutation of Arg106 in H‐FAPB (Prinsen & Veerkamp, 1996) or its replacement by Gln in rat I‐FABP (Jakoby et al ., 1993) markedly affects FA binding and conformational stability. The importance of Phe16 in ligand binding has been confirmed by studies on H‐FABP mutants (Prinsen & Veerkamp, 1996; Zimmerman et al ., 1999). All these conserved amino acid residues suggest that Ae ‐FABP's three‐dimensional structure may be similar to that of other FABPs.…”

New insights into intracellular lipid binding proteins: The role of buried water

“…For the F64S mutant, however, the only plausible explanation is a significant disruption of the hydrogen‐bonding network owing to the replacement of the Phe64 ring. Because FA binding is reduced 10% by the replacement of Phe64 (Zimmerman et al 1999), these results support the hypothesis that the ordered water molecules inside the H‐FABP binding pocket actually serve as a sort of hydration shell for the cluster of hydrophobic side‐chains at the bottom of the cavity (see Fig. 1) and simultaneously play a role in the ligand binding process.…”

“…In addition, a number of human H‐FABP mutants have been investigated by NMR spectroscopy at pH 7.0 and 298 K (Zimmerman et al 1999). All mutant protein spectra displayed the Ser82 OγH and His93 Nε2H resonances, except for the E72S and F64S mutants.…”

Probing the Interaction of Brain Fatty Acid Binding Protein (B-FABP) with Model Membranes

“…The cells were harvested by centrifugation. Purification was performed using an adapted protocol from previous works [57], [58]. Briefly, cell pellets were ressuspended in 20 mM phosphate buffer (pH 8.0), containing 0.1 M NaCl, 8 M urea, 1 mM dithiothreitol (DTT) and sonicated (Fischer Scientific, Waltham, MA, USA) in ice bath ten times for 30 seconds at 10% power with an intermittent 45 seconds interval to allow for sample cooling.…”