Analysis of the Two-State Behavior of the Thermal Unfolding of Serum Retinol Binding Protein Containing a Single Retinol Ligand

Academic Article

Abstract

  • Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 °C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 °C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (∆Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (∆HvH= 184 kcal/mol) and spectroscopic (∆HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR ⇌ U + R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N ⇌ U). Furthermore, the NR ⇌ U + R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR ⇌ U + R model is the simplest model that describes the thermal unfolding of this ligand-bound protein. Using an averaged van't Hoff enthalpy determined from fits of DSC and CD data, the cooperativity of this process was 0.925, indicating that the unfolding of the holoprotein is nearly two-state. © 1992, American Chemical Society. All rights reserved.
  • Digital Object Identifier (doi)

    Author List

  • Muccio DD; Waterhous DV; Fish F; Brouillette CG
  • Start Page

  • 5560
  • End Page

  • 5567
  • Volume

  • 31
  • Issue

  • 24