Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus.


Abstract

The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability toward thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV, and visible circular dichroism spectroscopy. The results show that the holo-protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo-protein of TtCuA were extremely stable toward unfolding by guanidine hydrochloride at ambient pH. The thermal unfolding studies at different values of pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different values of pH was analyzed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holo-protein showed that the variation of the unfolding free energy was associated with a pKa of approximately 5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein. Study holds ProTherm entries: 23862, 23863, 23864, 23865, 23866, 23867, 23868, 23869, 23870, 23871, 23872, 23873, 23874, 23875, 23876, 23877, 23878 Extra Details: oxidised holoprotein; CUA (copper center) domain dinuclear copper center; free energy of unfolding; holo-protein; apo-protein; protonation

Submission Details

ID: VZGXotc24

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Sujak A;Sanghamitra NJ;Maneg O;Ludwig B;Mazumdar S,Biophys. J. (2007) Thermostability of proteins: role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus. PMID:17604317
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
2LLN 2012-05-30 Solution structure of Thermus thermophilus apo-CuA
2FWL 2006-03-28 The cytochrome c552/CuA complex from Thermus thermophilus
2CUA 1999-05-28 1.6 THE CUA DOMAIN OF CYTOCHROME BA3 FROM THERMUS THERMOPHILUS
3S8G 2011-08-03 1.8 1.8 A structure of ba3 cytochrome c oxidase mutant (A120F) from Thermus thermophilus in lipid environment
3S8F 2011-08-03 1.8 1.8 A structure of ba3 cytochrome c oxidase from Thermus thermophilus in lipid environment
4G7S 2013-07-24 2.0 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236I from Thermus thermophilus
1XME 2005-03-22 2.3 Structure of Recombinant Cytochrome ba3 Oxidase from Thermus thermophilus
5U7N 2017-11-22 2.3 CRYSTAL STRUCTURE OF A CHIMERIC CUA DOMAIN (SUBUNIT II) OF CYTOCHROME BA3 FROM THERMUS THERMOPHILUS WITH THE AMICYANIN LOOP
5NDC 2017-08-23 2.3 Structure of ba3-type cytochrome c oxidase from Thermus thermophilus by serial femtosecond crystallography
1EHK 2001-02-22 2.4 CRYSTAL STRUCTURE OF THE ABERRANT BA3-CYTOCHROME-C OXIDASE FROM THERMUS THERMOPHILUS
4G70 2013-07-24 2.6 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236T from Thermus thermophilus
4G7Q 2013-07-24 2.6 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236L from Thermus thermophilus
4GP5 2013-01-16 2.7 Structure of Recombinant Cytochrome ba3 Oxidase mutant Y133W from Thermus thermophilus
3QJV 2012-01-25 2.8 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
4GP8 2013-01-16 2.8 Structure of Recombinant Cytochrome ba3 Oxidase mutant Y133W+T231F from Thermus thermophilus
3QJS 2012-01-25 2.8 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
3EH5 2009-04-21 2.8 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
4GP4 2013-01-16 2.8 Structure of Recombinant Cytochrome ba3 Oxidase mutant Y133F from Thermus thermophilus
4N4Y 2014-10-08 2.9 Structure of Recombinant Cytochrome ba3 Oxidase mutant G232V from Thermus thermophilus
2QPE 2007-12-11 2.9 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
3QJQ 2012-01-25 2.9 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
3EH4 2009-04-21 2.9 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
3QJU 2012-01-25 2.9 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
4G71 2013-07-24 2.9 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236N from Thermus thermophilus
3QJT 2012-01-25 2.95 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
4G7R 2013-07-24 3.05 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236A from Thermus thermophilus
3EH3 2009-04-21 3.1 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
4G72 2013-07-24 3.19 Structure of Recombinant Cytochrome ba3 Oxidase mutant V236M from Thermus thermophilus
3QJR 2012-01-25 3.2 The structure of and photolytic induced changes of carbon monoxide binding to the cytochrome ba3-oxidase from Thermus thermophilus
2QPD 2007-12-11 3.25 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
3S3B 2012-05-23 3.3 Structure of Thermus thermophilus cytochrome ba3 oxidase 240s after Xe depressurization
3BVD 2008-05-20 3.37 Structure of Surface-engineered Cytochrome ba3 Oxidase from Thermus thermophilus under Xenon Pressure, 100psi 5min
3S3D 2012-05-23 3.75 Structure of Thermus thermophilus cytochrome ba3 oxidase 480s after Xe depressurization
3S3C 2012-05-23 4.0 Structure of Thermus thermophilus cytochrome ba3 oxidase 360s after Xe depressurization
3S38 2012-05-23 4.2 Structure of Thermus thermophilus cytochrome ba3 oxidase 30s after Xe depressurization
3S3A 2012-05-23 4.25 Structure of Thermus thermophilus cytochrome ba3 oxidase 120s after Xe depressurization
3S33 2012-05-23 4.45 Structure of Thermus thermophilus cytochrome ba3 oxidase 10s after Xe depressurization
3S39 2012-05-23 4.8 Structure of Thermus thermophilus cytochrome ba3 oxidase 60s after Xe depressurization

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cytochrome c oxidase subunit 2 P98052 COX2_THETH
100.0 Cytochrome c oxidase subunit 2 Q5SJ80 COX2_THET8