Pro108 is important for folding and stabilization of adrenal ferredoxin, but does not influence the functional properties of the protein.
The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods. The reconstituted protein had spectroscopic, functional and redox properties similar to the Met-adrenodoxin-(4-108)-peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain. Rate of in vitro cluster insertion into the Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-adrenodoxin-(4-108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state. The Met-adrenodoxin-(4-107)-peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8 degrees C, compared with 51.7 degrees C for mutant Met-adrenodoxin-(4-108). The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.
Uhlmann
H
H
Fachbereich Pharmazie und Umwelttechnologie, Fachrichtung Biochemie, Universität des Saarlandes, Saarbrücken, Germany.
Iametti
S
S
Vecchio
G
G
Bonomi
F
F
Bernhardt
R
R
D000311:Adrenal Glands / Q000737:chemistry*; D000325:Adrenodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013053:Spectrophotometry; D013696:Temperature; D014508:Urea
D011994:Recombinant Proteins; D000325:Adrenodoxin; D014508:Urea; D011392:Proline