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
2CUA 1999-02-18T00:00:00+0000 1.6 THE CUA DOMAIN OF CYTOCHROME BA3 FROM THERMUS THERMOPHILUS
2FWL 2006-02-02T00:00:00+0000 0 The cytochrome c552/CuA complex from Thermus thermophilus
2LLN 2011-11-15T00:00:00+0000 0 Solution structure of Thermus thermophilus apo-CuA
1EHK 2000-02-21T00:00:00+0000 2.4 CRYSTAL STRUCTURE OF THE ABERRANT BA3-CYTOCHROME-C OXIDASE FROM THERMUS THERMOPHILUS
1XME 2004-10-01T00:00:00+0000 2.3 Structure of Recombinant Cytochrome ba3 Oxidase from Thermus thermophilus
2QPD 2007-07-23T00:00:00+0000 3.25 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
2QPE 2007-07-23T00:00:00+0000 2.9 An unexpected outcome of surface-engineering an integral membrane protein: Improved crystallization of cytochrome ba3 oxidase from Thermus thermophilus
3BVD 2008-01-07T00:00:00+0000 3.37 Structure of Surface-engineered Cytochrome ba3 Oxidase from Thermus thermophilus under Xenon Pressure, 100psi 5min
3EH3 2008-09-11T00:00:00+0000 3.1 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus
3EH4 2008-09-11T00:00:00+0000 2.9 Structure of the reduced form of cytochrome ba3 oxidase from Thermus thermophilus

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