Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in Escherichia coli.
SecA is an essential component of the protein secretory machinery of Escherichia coli. SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis. The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structures. It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation. SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution. Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol. It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.
Shinkai
A
A
Institute of Applied Microbiology, University of Tokyo, Japan.
Akita
M
M
Matsuyama
S
S
Mizushima
S
S
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D026901:Membrane Transport Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D006146:Guanidines; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D019791:Guanidine; D004229:Dithiothreitol