title: 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.
authors.affiliation: Department of Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands.
authors.forename: A W
authors.initials: AW
authors.lastname: Zimmerman
authors.forename: M
authors.initials: M
authors.lastname: Rademacher
authors.forename: H
authors.initials: H
authors.lastname: Rüterjans
authors.forename: C
authors.initials: C
authors.lastname: Lücke
authors.forename: J H
authors.initials: JH
authors.lastname: Veerkamp
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018996:Myelin P2 Protein / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry*; D009363:Neoplasm Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular; D019301:Oleic Acid / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D025521:Tumor Suppressor Proteins; D014508:Urea / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D019301:Oleic Acid; D010649:Phenylalanine; D014364:Tryptophan; D014508:Urea