Abstract
Pore-forming toxins are protein assemblies used by many organisms to disrupt the membranes of target cells. They are expressed as soluble monomers that assemble spontaneously into multimeric pores. However, owing to their complexity, the assembly processes have not been resolved in detail for any pore-forming toxin. To determine the assembly mechanism for the ring-shaped, homododecameric pore of the bacterial cytolytic toxin ClyA, we collected a diverse set of kinetic data using single-molecule spectroscopy and complementary techniques on timescales from milliseconds to hours, and from picomolar to micromolar ClyA concentrations. The entire range of experimental results can be explained quantitatively by a surprisingly simple mechanism. First, addition of the detergent n-dodecyl-β-D-maltopyranoside to the soluble monomers triggers the formation of assembly-competent toxin subunits, accompanied by the transient formation of a molten-globule-like intermediate. Then, all sterically compatible oligomers contribute to assembly, which greatly enhances the efficiency of pore formation compared with simple monomer addition.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cells, Cultured
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Erythrocytes / cytology
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Erythrocytes / drug effects
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Escherichia coli / genetics
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Escherichia coli / metabolism*
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Escherichia coli / pathogenicity
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Escherichia coli Proteins / biosynthesis
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / toxicity
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Fluorescence Resonance Energy Transfer
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Gene Expression
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Hemolysin Proteins / biosynthesis
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Hemolysin Proteins / chemistry*
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Hemolysin Proteins / genetics
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Hemolysin Proteins / toxicity
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Hemolysis / drug effects
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Horses
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Kinetics
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Maltose / analogs & derivatives
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Maltose / chemistry
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Models, Molecular
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Protein Multimerization*
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Protein Subunits / biosynthesis
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Protein Subunits / chemistry*
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Protein Subunits / genetics
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Protein Subunits / toxicity
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / toxicity
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Thermodynamics
Substances
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Escherichia coli Proteins
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Hemolysin Proteins
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Protein Subunits
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Recombinant Proteins
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dodecyl maltopyranoside
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hlyE protein, E coli
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Maltose