Secondary structure and orientation of the pore-forming toxin lysenin in a sphingomyelin-containing membrane.
Lysenin is a sphingomyelin-recognizing toxin which forms stable oligomers upon membrane binding and causes cell lysis. To get insight into the mechanism of the transition of lysenin from a soluble to a membrane-bound form, surface activity of the protein and its binding to lipid membranes were studied using tensiometric measurements, Fourier-transform infrared spectroscopy (FTIR) and FTIR-linear dichroism. The results showed cooperative adsorption of recombinant lysenin-His at the argon-water interface from the water subphase which suggested self-association of lysenin-His in solution. An assembly of premature oligomers by lysenin-His in solution was confirmed by blue native gel electrophoresis. When a monolayer composed of sphingomyelin and cholesterol was present at the interface, the rate of insertion of lysenin-His into the monolayer was considerably enhanced. Analysis of FTIR spectra of soluble lysenin-His demonstrated that the protein contained 27% beta-sheet, 28% aggregated beta-strands, 10% alpha-helix, 23% turns and loops and 12% different kinds of aggregated forms. In membrane-bound lysenin-His the total content of alpha-helices, turns and loops, and beta-structures did not change, however, the 1636cm(-1) beta-sheet band increased from 18% to 31% at the expense of the 1680cm(-1) beta-sheet structure. Spectral analysis of the amide I band showed that the alpha-helical component was oriented with at 41 degrees to the normal to the membrane, indicating that this protein segment could be anchored in the hydrophobic core of the membrane.
Hereć
Monika
M
Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
Gagoś
Mariusz
M
Kulma
Magdalena
M
Kwiatkowska
Katarzyna
K
Sobota
Andrzej
A
Gruszecki
Wiesław I
WI
D000327:Adsorption; D000388:Air; D006639:Histidine; D008051:Lipid Bilayers / Q000737:chemistry*; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry*; D011312:Pressure; D017433:Protein Structure, Secondary; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D013109:Sphingomyelins / Q000737:chemistry*; D013997:Time Factors; D014118:Toxins, Biological / Q000737:chemistry*; D014867:Water
D008051:Lipid Bilayers; D052899:Pore Forming Cytotoxic Proteins; D013109:Sphingomyelins; D014118:Toxins, Biological; C107040:lysenin; D014867:Water; D006639:Histidine