Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate.
During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.
Rausell
Carolina
C
Departamento de Microbiología Molecular and Departamento de Reconocimiento Molecular y Bioestructura, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo postal 510-3, Cuernavaca 62250, Morelos, Mexico.
Muñoz-Garay
Carlos
C
Miranda-CassoLuengo
Raúl
R
Gómez
Isabel
I
Rudiño-Piñera
Enrique
E
Soberón
Mario
M
Bravo
Alejandra
A
D020106:Acrylamide / Q000737:chemistry; D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism*; D006460:Hemolysin Proteins; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000032:analysis / Q000737:chemistry*; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*; D014867:Water / Q000737:chemistry
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D012996:Solutions; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014867:Water; D020106:Acrylamide; D014364:Tryptophan