Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfur-transferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH- cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.
|Titolo:||GroEL-assisted refolding of adrenodoxin during chemical cluster insertion|
|Autori interni:||BONOMI, FRANCESCO (Ultimo)|
IAMETTI, STEFANIA (Primo)
|Parole Chiave:||Adrenodoxin; Chaperones; Iron-sulfur clusters; Protein refolding|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2001|
|Appare nelle tipologie:||01 - Articolo su periodico|