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  • The EMBO Journal (2003) 22, 4968 - 4979
  • doi:10.1093/emboj/cdg498

Single-molecule imaging of cooperative assembly of big gamma-hemolysin on erythrocyte membranes

Vananh T. Nguyen1, Yoshiyuki Kamio1,2 and Hideo Higuchi2,3

  1. Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
  2. Center for Interdisciplinary Research, Sendai 980-8579, Japan
  3. Department of Metallurgy, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan

Correspondence to:

Hideo Higuchi, E-mail: higuchi@material.tohoku.ac.jp

Received 19 May 2003; Accepted 11 August 2003; Revised 8 August 2003

Single-molecule fluorescence imaging was used to investigate assembly of Staphylococcus aureus LukF and HS monomers into pore-forming oligomers (gamma-hemolysin) on erythrocyte membranes. We distinguished the hetero-oligomers from the monomers, as indicated by fluorescence resonance energy transfer between different dyes attached to monomeric subunits. The stoichiometry of LukF (donor) and HS (acceptor) subunits in oligomers was deduced from the acceptor emission intensities during energy transfer and by direct acceptor excitation, respectively. Based on populations of monomeric and oligomeric intermediates, we estimated 11 sequential equilibrium constants for the assembly pathway, beginning with membrane binding of monomers, proceeding through single pore oligomerization, and culminating in the formation of clusters of pores. Several stages are highly cooperative, critically enhancing the efficiency of assembly.

  • Keywords:

    • association constants,
    • cell membranes,
    • oligomeric intermediates,
    • pore assembly,
    • single-FRET