Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA.
The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.
Zhang
F
F
Division of Molecular and Structural Biology, Ontario Cancer Institute, Canada.
Yin
Y
Y
Arrowsmith
C H
CH
Ling
V
V
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D016978:Mannheimia haemolytica / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C018579:leukotoxin; D005982:Glutathione Transferase