title: The interrelationship between ligand binding and self-association of the folate binding protein. The role of detergent-tryptophan interaction.
abstract: BACKGROUND
  The folate binding protein (FBP) regulates homeostasis and intracellular trafficking of folic acid, a vitamin of decisive importance in cell division and growth. We analyzed whether interrelationship between ligand binding and self-association of FBP plays a significant role in the physiology of folate binding.
  
  
  METHODS
  Self-association behavior of apo- and holo-FBP was addressed through size exclusion chromatography, SDS-PAGE, mass spectrometry, surface plasmon resonance and fluorescence spectroscopy.
  
  
  RESULTS
  Especially holo-FBP exhibits concentration-dependent self-association at pH 7.4 (pI), and is more prone to associate into stable complexes than apo-FBP. Even more pronounced was the tendency to complexation between apo-FBP and holo-FBP in accord with a model predicting association between apo and holo monomers [19]. This will lead to removal of apo monomers from the reaction scheme resulting in a weak incomplete ligand binding similar to that observed at FBP concentrations <10nM. The presence of synthetic and natural detergents normalized folate binding kinetics and resulted in appearance of monomeric holo-FBP. Fluorescence spectroscopy indicated molecular interactions between detergent and tryptophan residues located in hydrophobic structures of apo-FBP which may participate in protein associations.
  
  
  CONCLUSIONS
  Self-association into multimers may protect binding sites, and in case of holo-FBP even folate from biological degradation. High-affinity folate binding in body secretions, typically containing 1-10nM FBP, requires the presence of natural detergents, i.e. cholesterol and phospholipids, to avoid complexation between apo- and holo-FBP.
authors.affiliation: Department of Clinical Biochemistry, Hillerød Hospital, Dyrehavevej 29, DK-3400 Hillerød, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Holm
authors.forename: Christian
authors.initials: C
authors.lastname: Schou
authors.forename: Linnea N
authors.initials: LN
authors.lastname: Babol
authors.forename: Anders J
authors.initials: AJ
authors.lastname: Lawaetz
authors.forename: Susanne W
authors.initials: SW
authors.lastname: Bruun
authors.forename: Morten Z
authors.initials: MZ
authors.lastname: Hansen
authors.forename: Steen I
authors.initials: SI
authors.lastname: Hansen
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D003902:Detergents / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005492:Folic Acid / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D003902:Detergents; D008024:Ligands; D014364:Tryptophan; D005492:Folic Acid