Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen.


Abstract

The thermal denaturation of the cupredoxin plastocyanin (PC) from spinach has been studied with the aim of improving the understanding of factors involved in the conformational stability of antiparallel beta-sheet proteins. Studies using differential scanning calorimetry have been complemented with nuclear magnetic resonance spectroscopy, absorbance spectroscopy, dynamic light scattering, and mass spectrometry in elucidation of the effect of the copper-site oxidation state on the irreversible thermal denaturation process. Our results indicate that copper-catalyzed oxidation of the metal-ligating cysteine is the sole factor resulting in thermal irreversibility. However, this can be prevented in reduced protein by the removal of molecular oxygen. Application of a two-state equilibrium transition model to the folding process thus allowed the extraction of thermodynamic parameters for the reduced protein (Delta(trs)H = 494 kJ mol(-1), DeltaH(vH) = 343 kJ mol(-1), and T(m) = 71 degrees C). However, anaerobically denatured oxidized protein and all aerobically denatured species undergo covalent modification as a result of the copper-catalyzed oxidation of the metal-ligating cysteine residue resulting in the formation of both oxidized monomers and disulfide-linked dimers. On the basis of these results, a general mechanism for the irreversible thermal denaturation of cupredoxins is proposed. The results presented here also indicate that PC, as opposed to the previously characterized homologous protein azurin, unfolds via at least one significantly populated intermediate state (DeltaH(vH)/Delta(trs)H = 0.7) despite the almost identical native state topologies of these proteins. These findings will aid the characterization of the stability of PC and other cupredoxins and possibly of all cysteine-ligating metal-binding proteins. Study holds ProTherm entries: 16548, 16549, 16550, 16551, 16552 Extra Details: reduced, aerobic conformational stability; antiparallel beta-sheet; metal-ligating cysteine; copper-catalyzed oxidation

Submission Details

ID: BvtrXMdK

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Sandberg A;Harrison DJ;Karlsson BG,Biochemistry (2003) Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen. PMID:12939160
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
2PCF 1998-04-08 THE COMPLEX OF CYTOCHROME F AND PLASTOCYANIN DETERMINED WITH PARAMAGNETIC NMR. BASED ON THE STRUCTURES OF CYTOCHROME F AND PLASTOCYANIN, 10 STRUCTURES
1YLB 2005-04-05 NMR solution structure of the reduced spinach plastocyanin
1AG6 1998-10-21 1.6 PLASTOCYANIN FROM SPINACH
1TEF 2005-11-01 1.9 Crystal structure of the spinach plastocyanin mutants G8D/K30C/T69C and K30C/T69C- a study of the effect on crystal packing and thermostability from the introduction of a novel disulfide bond
1TEG 2005-11-01 1.96 Crystal structure of the spinach plastocyanin mutants G8D/K30C/T69C and K30C/T69C- a study of the effect on crystal packing and thermostability from the introduction of a novel disulfide bond
1OOW 2004-02-17 2.0 The crystal structure of the spinach plastocyanin double mutant G8D/L12E gives insight into its low reactivity towards photosystem 1 and cytochrome f

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Plastocyanin, chloroplastic P00289 PLAS_SPIOL