title: The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH.
abstract: The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH7.4 (pI=7-8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3μM holo-FBP showed Tm (76°C) and molar enthalpy (146kcalM(-1)) values increasing to 78°C and 163kcalM(-1) at 10μM holo-FBP, while those of apo-FBP were 55°C and 105kcalM(-1). Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10μM) in DLS after a step-wise rise in temperature to 78°C≈Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH5 (Tm 38°C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.
authors.affiliation: Department of Clinical Biochemistry, Nordsjællands Hospital-Hillerød, University Hospital Copenhagen, Dyrehavevej 29, DK-3400 Hillerød, Denmark. Electronic address: Janf@holm.mail.dk.
authors.forename: Jan
authors.initials: J
authors.lastname: Holm
authors.affiliation: GE Healthcare, Life Sciences Bio-Sciences AB, Bjørkgatan 30, 751 84 Uppsala, Sweden.
authors.forename: Linnea N
authors.initials: LN
authors.lastname: Babol
authors.affiliation: GE Healthcare, Life Sciences Bio-Sciences AB, Bjørkgatan 30, 751 84 Uppsala, Sweden.
authors.forename: Natalia
authors.initials: N
authors.lastname: Markova
authors.affiliation: Faculty of Science, Department of Food Science, Spectroscopy and Chemometrics, University of Copenhagen, Rolighedsvej 26, DK-1958 Frederiksberg, Denmark.
authors.forename: Anders J
authors.initials: AJ
authors.lastname: Lawaetz
authors.affiliation: Department of Clinical Biochemistry, Nordsjællands Hospital-Hillerød, University Hospital Copenhagen, Dyrehavevej 29, DK-3400 Hillerød, Denmark. Electronic address: Steen.Ingemann.Hansen@Regionh.dk.
authors.forename: Steen I
authors.initials: SI
authors.lastname: Hansen
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D005492:Folic Acid / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D011485:Protein Binding; D055550:Protein Stability; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence
chemical_list: D002352:Carrier Proteins; D008024:Ligands; D005492:Folic Acid
keywords: Differential scanning calorimetry; Dynamic light scattering; Fluorescence changes associated with irreversible thermal unfolding; Folate binding protein (soluble folate receptor); Temperature-dependent self-association; Thermoresistant holo-protein