The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

• Published in: Biochimica et biophysica acta Vol. 1844; no. 3; pp. 512 - 519
• Main Authors: Holm, Jan, Babol, Linnea N., Markova, Natalia, Lawaetz, Anders J., Hansen, Steen I.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.03.2014
• Subjects: Animals; Calorimetry, Differential Scanning; Carrier Proteins - metabolism; Cattle; Differential scanning calorimetry; Dynamic light scattering; Fluorescence changes associated with irreversible thermal unfolding; Folate binding protein (soluble folate receptor); Folic Acid - metabolism; Hot Temperature; Hydrogen-Ion Concentration; Ligands; Protein Binding; Protein Stability; Protein Unfolding; Spectrometry, Fluorescence; Temperature-dependent self-association; Thermoresistant holo-protein; Temperature-dependent self-association; Differential scanning calorimetry; Folate binding protein (soluble folate receptor); Dynamic light scattering; Thermoresistant holo-protein; Fluorescence changes associated with irreversible thermal unfolding
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2013.12.009

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. ●Holo-FBP multimers are less prone to dissociate upon heating than apo-FBP multimers.●Folate binding increases thermal stability of FBP drastically at neutral pH (pI=7–8).●Multimerization and thermostability are increased at a higher holo-FBP concentration.●At pH5 cationic FBP is thermolabile and unfolds unless NaCl is added.●Weak-affinity folate binding persists after irreversible thermal unfolding of FBP.