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
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1KT6 2003-06-03 1.1 Crystal structure of bovine holo-RBP at pH 9.0
1KT7 2003-06-03 1.27 Crystal structure of bovine holo-RBP at pH 7.0
1KT3 2003-06-03 1.4 Crystal structure of bovine holo-RBP at pH 2.0
1KT5 2003-06-03 1.46 Crystal structure of bovine holo-RBP at pH 4.0
1KT4 2003-06-03 1.46 Crystal structure of bovine holo-RBP at pH 3.0
5NU2 2018-03-07 1.5 Structure of non-fluorescent human urine RBP4
5NU7 2018-03-07 1.5 Structure of human holo plasma RBP4
5NU9 2018-03-07 1.5 Structure of human amniotic fluid RBP4 saturated with palmitate
5NU8 2018-03-07 1.59 Structure of human urine RBP4 saturated with palmitate
5NUB 2018-03-14 1.6 Structure of human amniotic fluid RBP4 saturated with laurate
5NUA 2018-03-07 1.6 Structure of human urine RBP4 saturated with laurate
1AQB 1998-01-28 1.65 RETINOL-BINDING PROTEIN (RBP) FROM PIG PLASMA
2WQ9 2010-09-01 1.65 Crystal Structure of RBP4 bound to Oleic Acid
5NU6 2018-03-07 1.68 Structure of non-fluorescent human amniotic fluid RBP4
1JYD 2003-07-01 1.7 Crystal Structure of Recombinant Human Serum Retinol-Binding Protein at 1.7 A Resolution
1HBQ 1994-01-31 1.7 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1FEL 1994-11-01 1.8 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
2WR6 2010-09-15 1.8 Structure of the complex of RBP4 with linoleic acid
1FEN 1994-11-01 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
1HBP 1994-01-31 1.9 CRYSTAL STRUCTURE OF LIGANDED AND UNLIGANDED FORMS OF BOVINE PLASMA RETINOL-BINDING PROTEIN
1ERB 1994-01-31 1.9 THE INTERACTION OF N-ETHYL RETINAMIDE WITH PLASMA RETINOL-BINDING PROTEIN (RBP) AND THE CRYSTAL STRUCTURE OF THE RETINOID-RBP COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1FEM 1994-11-01 1.9 CRYSTALLOGRAPHIC STUDIES ON COMPLEXES BETWEEN RETINOIDS AND PLASMA RETINOL-BINDING PROTEIN
5NTY 2018-03-07 2.0 Structure of non-fluorescent human plasma RBP4
1JYJ 2003-07-01 2.0 Crystal Structure of a Double Variant (W67L/W91H) of Recombinant Human Serum Retinol-binding Protein at 2.0 A Resolution
1RBP 1991-07-15 2.0 CRYSTALLOGRAPHIC REFINEMENT OF HUMAN SERUM RETINOL BINDING PROTEIN AT 2 ANGSTROMS RESOLUTION
4O9S 2014-07-02 2.3 Crystal structure of Retinol-Binding Protein 4 (RBP4)in complex with a non-retinoid ligand
4PSQ 2014-07-02 2.4 Crystal Structure of Retinol-Binding Protein 4 (RBP4) in complex with a non-retinoid ligand
1BRP 1994-01-31 2.5 CRYSTAL STRUCTURE OF THE TRIGONAL FORM OF HUMAN PLASMA RETINOL-BINDING PROTEIN AT 2.5 ANGSTROMS RESOLUTION
1BRQ 1994-01-31 2.5 CRYSTAL STRUCTURE OF THE TRIGONAL FORM OF HUMAN PLASMA RETINOL-BINDING PROTEIN AT 2.5 ANGSTROMS RESOLUTION
2WQA 2010-09-01 2.85 Complex of TTR and RBP4 and Oleic Acid
3FMZ 2009-01-27 2.9 Crystal Structure of Retinol-Binding Protein 4 (RBP4) in complex with non-retinoid ligand
1RLB 1996-04-11 3.1 RETINOL BINDING PROTEIN COMPLEXED WITH TRANSTHYRETIN
1QAB 1999-02-16 3.2 The structure of human retinol binding protein with its carrier protein transthyretin reveals interaction with the carboxy terminus of RBP
3BSZ 2008-11-11 3.38 Crystal structure of the transthyretin-retinol binding protein-Fab complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.8 Retinol-binding protein 4 P06912 RET4_RABIT
92.3 Retinol-binding protein 4 P18902 RET4_BOVIN
92.9 Retinol-binding protein 4 P27485 RET4_PIG
94.5 Retinol-binding protein 4 Q28369 RET4_HORSE
94.5 Retinol-binding protein 4 M5AXY1 RET4_FELCA
100.0 Retinol-binding protein 4 P61641 RET4_PANTR
100.0 Retinol-binding protein 4 P02753 RET4_HUMAN