Impact of the presequence of a mitochondrium-targeted precursor, preadrenodoxin, on folding, catalytic activity, and stability of the protein in vitro.
Bovine preadrenodoxin, an adrenocortical precursor protein destined for mitochondrial import, was expressed in Escherichia coli as an [2Fe-2S] cluster-containing protein. It was found in inclusion bodies, purified from there, and finally reconstituted to obtain soluble holo-protein. The impact of the presequence on folding of the protein using biochemical and biophysical approaches has been investigated. Upon unfolding the preprotein reveals a decrease in the denaturational enthalpy and heat capacity compared with mature adrenodoxin, indicating an incomplete unfolding of the preprotein with remaining residual structure. Moreover, the data obtained show that the presequence is solvent exposed in aqueous solution with no preference for secondary structure elements and that it does not disturb the accurate folding of the mature part of the protein. The latter conclusion is also based on the finding that the precursor in vitro exhibits electron transfer function comparable to the mature protein, adrenodoxin. While the reduction of cytochrome c, reflecting the interaction between adrenodoxin and its reductase, and the interaction with CYP11B1 have not been significantly affected by the presence of the presequence, the binding affinity of preadrenodoxin to CYP11A1 is 5.5-fold lower than that of the mature form.
Goder
V
V
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, Berlin-Buch, D-13125, Germany.
Beckert
V
V
Pfeil
W
W
Bernhardt
R
R
D000325:Adrenodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C044938:preadrenodoxin; D000325:Adrenodoxin; D010447:Peptide Hydrolases