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

作者：Holm Jan; Babol Linnea N; Markova Natalia; Lawaetz Anders J; Hansen Steen I^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2014, 1844(3): 512-519.

DOI：10.1016/j.bbapap.2013.12.009

• Folate binding protein (soluble folate receptor)
• Temperature-dependent self-association
• Dynamic light scattering
• Thermoresistant holo-protein
• Differential scanning calorimetry
• Fluorescence changes associated with irreversible thermal unfolding

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 pH 7.4 (pl = 7-8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 mu M holo-FBP showed Tm (76 degrees C) and molar enthalpy (146 kcal M-1) values increasing to 78 C and 163 kcal M-1 at 10 mu M holo-FBP, while those of apo-FBP were 55 C and 105 kcal M-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 (10 mu M) 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 pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M 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.

• 出版日期2014-3