Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli.
PsbO protein is an important constituent of the water-oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond. The PsbO protein purified from a pET32 system that includes thioredoxin fusion is properly folded and functionally active. Urea unfolding experiments imply that the reduction of the single disulfide bridge decreases stability of the protein. Analysis of inter-residue contact density through the PsbO molecule shows that Cys51 is located in a cluster with high contact density. Reduction of the Cys28-Cys51 bond is proposed to perturb the packing interactions in this cluster and destabilize the protein as a whole. Taken together, our results give evidence that PsbO exists in solution as a compact highly ordered structure, provided that the disulfide bridge is not reduced.
Nikitina
Julia
J
Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 901 87 Umea, Sweden.
Shutova
Tatiana
T
Melnik
Bogdan
B
Chernyshov
Sergey
S
Marchenkov
Victor
V
Semisotnov
Gennady
G
Klimov
Vyacheslav
V
Samuelsson
Göran
G
D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D018724:Spinacia oleracea / Q000737:chemistry; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins; C054266:photosystem II manganese-stabilizing protein; D013879:Thioredoxins