Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.


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 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta 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 (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with 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 in equilibrium with U). Furthermore, the NR in equilibrium with 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 in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 5309, 5310 Extra Details: ligand-bound protein; two-state thermodynamic model;,ligand dissociation; holoprotein

Submission Details

ID: 4ES7e3633

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Muccio DD;Waterhous DV;Fish F;Brouillette CG,Biochemistry (1992) Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand. PMID:1610801
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