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 (Phe4, Trp8 and Phe64), portal region (Phe16), hinge region (Leu66, Gly67), second portal region (Glu72) and at the protein surface (Lys21) respectively. Oleic acid-binding affinity and conformational stability of human H-FABP are significantly decreased or completely lost by mutation of Trp8 or Phe16. NMR spectra confirmed that these residues are important for the stability of the protein fold. Substitution of Phe4 or Phe64 resulted in less stability, but oleic acid-binding affinity was not affected. Mutation of Lys21 had no effect on either structural integrity or fatty acid-binding affinity. Replacement of Leu66 or Gly67 did not affect fatty acid binding, but protein stability was reduced. Finally, mutation of Glu72 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.
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December 1999
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Research Article|
November 24 1999
Functional and conformational characterization of new mutants of heart fatty acid-binding protein
Aukje W. ZIMMERMAN;
Aukje W. ZIMMERMAN
*Department of Biochemistry, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
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Martin RADEMACHER;
Martin RADEMACHER
†Institut für Biophysikalische Chemie, J. W. Goethe-Universität Frankfurt, Germany
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Heinz RüTERJANS;
Heinz RüTERJANS
†Institut für Biophysikalische Chemie, J. W. Goethe-Universität Frankfurt, Germany
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Christian LüCKE;
Christian LüCKE
†Institut für Biophysikalische Chemie, J. W. Goethe-Universität Frankfurt, Germany
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Jacques H. VEERKAMP
Jacques H. VEERKAMP
1
*Department of Biochemistry, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
1To whom correspondence should be addressed (e-mail J.Veerkamp@bioch.kun.nl).
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Publisher: Portland Press Ltd
Received:
June 16 1999
Revision Received:
July 29 1999
Accepted:
September 08 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 344 (2): 495–501.
Article history
Received:
June 16 1999
Revision Received:
July 29 1999
Accepted:
September 08 1999
Citation
Aukje W. ZIMMERMAN, Martin RADEMACHER, Heinz RüTERJANS, Christian LüCKE, Jacques H. VEERKAMP; Functional and conformational characterization of new mutants of heart fatty acid-binding protein. Biochem J 1 December 1999; 344 (2): 495–501. doi: https://doi.org/10.1042/bj3440495
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