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Cellular Fatty Acid-binding Proteins pp 81–93Cite as

Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction

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Part of the book series: Developments in Molecular and Cellular Biochemistry ((DMCB,volume 6))

Abstract

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE+ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a supE- strain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel β-strands organized into two orthogonally situated β-sheets. The overall conformation of the protein resembles that of a clam — hence the term β-clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg106 and Gin115. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABF has a similar overall conformation to holo-I-FABP indicating that the β-clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations of several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism.

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Authors and Affiliations

  1. Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, NY, 10462, USA

    James C. Sacchettini

  2. Department of Biochemistry, University of Minnesota, Minneapolis, MN, 55455, USA

    Leonard J. Banaszak

  3. Dept. of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Box 8231, St. Louis, MO, 63110, USA

    Jeffrey I. Gordon

Authors
  1. James C. Sacchettini
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  2. Leonard J. Banaszak
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  3. Jeffrey I. Gordon
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Editors and Affiliations

  1. Department of Physiology, University of Limburg, Maastricht, The Netherlands

    Jan F. C. Glatz  & Ger J. Van Der Vusse  & 

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Sacchettini, J.C., Banaszak, L.J., Gordon, J.I. (1990). Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction. In: Glatz, J.F.C., Van Der Vusse, G.J. (eds) Cellular Fatty Acid-binding Proteins. Developments in Molecular and Cellular Biochemistry, vol 6. Springer, Boston, MA. https://doi.org/10.1007/978-1-4615-3936-0_11

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  • DOI: https://doi.org/10.1007/978-1-4615-3936-0_11

  • Publisher Name: Springer, Boston, MA

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