Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride.


Abstract

Isothermal guanidine hydrochloride (GdnHCl)-induced denaturation curves obtained at 14 different temperatures in the range 273-323 K have been used in conjunction with thermally-induced denaturation curves obtained in the presence of 15 different concentrations of GdnHCl to characterize the thermodynamics of cold and heat denaturation of barstar. The linear free energy model has been used to determine the excess changes in free energy, enthalpy, entropy, and heat capacity that occur on denaturation. The stability of barstar in water decreases as the temperature is decreased from 300 to 273 K. This decrease in stability is not accompanied by a change in structure as monitored by measurement of the mean residue ellipticities at both 222 and 275 nm. When GdnHCl is present at concentrations between 1.2 and 2.0 M, the decrease in stability with decrease in temperature is however so large that the protein undergoes cold denaturation. The structural transition accompanying the cold denaturation process has been monitored by measuring the mean residue ellipticity at 222 nm. The temperature dependence of the change in free energy, obtained in the presence of 10 different concentrations of GdnHCl in the range 0.2-2.0 M, shows a decrease in stability with a decrease as well as an increase in temperature from 300 K. Values of the thermodynamic parameters governing the cold and the heart denaturation of barstar have been obtained with high precision by analysis of these bell-shaped stability curves. The change in heat capacity accompanying the unfolding reaction, delta Cp, has a value of 1460 +/- 70 cal mol-1 K-1 in water. The dependencies of the changes in enthalpy, entropy, free energy, and heat capacity on GdnHCl concentration have been analyzed on the basis of the linear free energy model. The changes in enthalpy (delta Hi) and entropy (delta Si), which occur on preferential binding of GdnHCl to the unfolded state, vis-a-vis the folded state, both have a negative value at low temperatures. With an increase in temperature delta Hi makes a less favorable contribution, while delta Si makes a more favorable contribution to the change in free energy (delta Gi) due to this interaction. The change in heat capacity (delta CPi) that occurs on preferential interaction of GdnHCl with the unfolded form has a value of only 53 +/- 36 cal mol-1 K-1 M-1. The data validate the linear free energy model that is commonly used to analyze protein stability. Study holds ProTherm entries: 4756 Extra Details: additive : EDTA(100 uM), linear free energy model; cold denaturation process;,bell-shaped stability curves; protein stability

Submission Details

ID: fYGWzrHr

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Agashe VR;Udgaonkar JB,Biochemistry (1995) Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride. PMID:7880824
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
1BTA 1994-07-31 THREE-DIMENSIONAL SOLUTION STRUCTURE AND 13C ASSIGNMENTS OF BARSTAR USING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1L1K 2002-12-04 NMR Identification and Characterization of the Flexible Regions in the 160 KD Molten Globule-like Aggregate of Barstar at Low pH
1BTB 1994-07-31 THREE-DIMENSIONAL SOLUTION STRUCTURE AND 13C ASSIGNMENTS OF BARSTAR USING NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
1AB7 1997-09-04 NMR 15N RELAXATION AND STRUCTURAL STUDIES REVEAL CONFORMATIONAL EXCHANGE IN BARSTAR C40/82A, 30 STRUCTURES
2ZA4 2008-05-20 1.58 Crystal Structural Analysis of Barnase-barstar Complex
1AY7 1999-03-02 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
1B2S 1998-12-08 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1X1Y 2005-04-26 1.9 Water-mediate interaction at aprotein-protein interface
2HXX 2006-08-22 2.0 Aminotryptophan Barstar
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
1B27 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-protein interface
1B2U 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
3DA7 2009-04-14 2.25 A conformationally strained, circular permutant of barnase
1X1U 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1X1X 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1B3S 1998-12-09 2.39 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BGS 1994-04-30 2.6 RECOGNITION BETWEEN A BACTERIAL RIBONUCLEASE, BARNASE, AND ITS NATURAL INHIBITOR, BARSTAR
1A19 1998-04-08 2.76 BARSTAR (FREE), C82A MUTANT

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Barstar P11540 BARS_BACAM