Antifolding activity of the SecB chaperone is essential for secretion of HasA, a quickly folding ABC pathway substrate.
We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s(-1)) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s(-1)) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.
Wolff
Nicolas
N
Unité de Résonance Magnétique Nucléaire des Biomolécules, Institut Pasteur, Paris, France.
Sapriel
Guillaume
G
Bodenreider
Christophe
C
Chaffotte
Alain
A
Delepelaire
Philippe
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D015233:Models, Statistical; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012706:Serratia marcescens / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D002352:Carrier Proteins; C093956:HasA protein, Serratia marcescens; D008565:Membrane Proteins; C057842:SecB protein, Bacteria; D000255:Adenosine Triphosphate; D019791:Guanidine