Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies.


Abstract

The thermodynamic stability and temperature induced structural changes of oxidized thioredoxin h from Chlamydomonas reinhardtii have been studied using differential scanning calorimetry (DSC), near- and far-UV circular dichroism (CD), and fluorescence spectroscopies. At neutral pH, the heat induced unfolding of thioredoxin h is irreversible. The irreversibly unfolded protein is unable to refold due to the formation of soluble high-order oligomers. In contrast, at acidic pH the heat induced unfolding of thioredoxin h is fully reversible and thus allows the thermodynamic stability of this protein to be characterized. Analysis of the heat induced unfolding at acidic pH using calorimetric and spectroscopic methods shows that the heat induced denaturation of thioredoxin h can be well approximated by a two-state transition. The unfolding of thioredoxin h is accompanied by a large heat capacity change [6.0 +/- 1.0 kJ/(mol.K)], suggesting that at low pH a cold denaturation should be observed at the above-freezing temperatures for this protein. All used methods (DSC, near-UV CD, far-UV CD, Trp fluorescence) do indeed show that thioredoxin h undergoes cold denaturation at pH <2.5. The cold denaturation of thioredoxin h cannot, however, be fitted to a two-state model of unfolding. Furthermore, according to the far-UV CD, thioredoxin h is fully unfolded at pH 2.0 and 0 degrees C, whereas the other three methods (near-UV CD, fluorescence, and DSC) indicate that under these conditions 20-30% of the protein molecules are still in the native state. Several alternative mechanisms explaining these results such as structural differences in the heat and cold denatured state ensembles and the two-domain structure of thioredoxin h are discussed. Study holds ProTherm entries: 10202, 10203, 10204, 10205, 10206, 10207, 10208, 10209, 10210, 10211, 10212, 10213, 10214, 10215, 10216, 10217, 10218 Extra Details: thermodynamic stability; spectroscopic methods;,two-state transition; two-domain structure

Submission Details

ID: NNqyZHXU3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:39 p.m.

Version: 1

Publication Details
Richardson JM;Lemaire SD;Jacquot JP;Makhatadze GI,Biochemistry (2000) Difference in the mechanisms of the cold and heat induced unfolding of thioredoxin h from Chlamydomonas reinhardtii: spectroscopic and calorimetric studies. PMID:10998255
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
1TOF 1996-12-07 THIOREDOXIN H (OXIDIZED FORM), NMR, 23 STRUCTURES
6Q6T 2019-01-16 0.94 Crystal structure (orthorombic form) of C36S mutant of thioredoxin h1 from Chlamydomonas reinhardtii
6Q6V 2019-01-16 1.22 Crystal structure (trigonal form) of C36S mutant of thioredoxin h1 from Chlamydomonas reinhardtii
6I19 2018-12-05 1.38 Crystal structure of Chlamydomonas reinhardtii thioredoxin h1
6Q47 2019-01-16 1.57 Crystal structure of partially oxidized thioredoxin h1 from Chlamydomonas reinhardtii
6Q46 2019-01-16 1.7 Crystal structure of reduced thioredoxin h1 from Chlamydomonas reinhardtii
6Q6U 2019-01-16 1.81 Crystal structure of C39S mutant of thioredoxin h1 from Chlamydomonas reinhardtii
1EP7 2001-12-12 2.1 CRYSTAL STRUCTURE OF WT THIOREDOXIN H FROM CHLAMYDOMONAS REINHARDTII
1EP8 2001-12-12 2.2 CRYSTAL STRUCTURE OF A MUTATED THIOREDOXIN, D30A, FROM CHLAMYDOMONAS REINHARDTII

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Thioredoxin H-type P80028 TRXH_CHLRE