TY - JOUR
T1 - The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH
AU - Holm, Jan
AU - Babol, Linnea N.
AU - Markova, Natalia
AU - Lawaetz, Anders Juul
AU - Hansen, Steen I.
PY - 2014/3
Y1 - 2014/3
N2 - 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 (pI = 7-8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 C) and molar enthalpy (146 kcal M- 1) values increasing to 78 C and 163 kcal M- 1 at 10 μM 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 μM) 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.
AB - 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 (pI = 7-8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 C) and molar enthalpy (146 kcal M- 1) values increasing to 78 C and 163 kcal M- 1 at 10 μM 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 μM) 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.
U2 - 10.1016/j.bbapap.2013.12.009
DO - 10.1016/j.bbapap.2013.12.009
M3 - Journal article
SN - 1570-9639
VL - 1844
SP - 512
EP - 519
JO - B B A - Proteins and Proteomics
JF - B B A - Proteins and Proteomics
IS - 3
ER -