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

  • James C. Sacchettini
  • Leonard J. Banaszak
  • Jeffrey I. Gordon
Part of the Developments in Molecular and Cellular Biochemistry book series (DMCB, volume 6)


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.

Key words

fatty acid binding protein prokaryotic expression vectors x-ray crystallography 


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Copyright information

© Springer Science+Business Media Dordrecht 1990

Authors and Affiliations

  • James C. Sacchettini
    • 1
  • Leonard J. Banaszak
    • 2
  • Jeffrey I. Gordon
    • 3
  1. 1.Department of Biochemistry, Albert Einstein College of MedicineYeshiva UniversityNew YorkUSA
  2. 2.Department of BiochemistryUniversity of MinnesotaMinneapolisUSA
  3. 3.Dept. of Biochemistry & Molecular BiophysicsWashington University School of MedicineSt. LouisUSA

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