Dimerization stabilizes the pore-forming toxin aerolysin in solution.
Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila. Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate. Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism. This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation. However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected. Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer. Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin. These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur.
van der Goot
F G
FG
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Ausio
J
J
Wong
K R
KR
Pattus
F
F
Buckley
J T
JT
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D001427:Bacterial Toxins / Q000737:chemistry*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004120:Dimethyl Suberimidate; D004355:Drug Stability; D005456:Fluorescent Dyes; D007473:Ion Channels; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011498:Protein Precursors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000817:Anilino Naphthalenesulfonates; D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D007473:Ion Channels; D046911:Macromolecular Substances; D052899:Pore Forming Cytotoxic Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D012996:Solutions; D004120:Dimethyl Suberimidate; C032676:aerolysin; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan