Single-molecule visualization of environment-sensitive fluorophores inserted into cell membranes by staphylococcal gamma-hemolysin.
Single-molecule imaging of the entrance of a protein into the hydrophobic environment of a cell membrane was investigated. The pre-stem of LukF, one of the two components of the pore-forming toxin staphylococcal gamma-hemolysin, was specifically labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (Badan), an environment-sensitive fluorophore. Incubation of this derivative with erythrocyte ghost membranes resulted in a pronounced increase in fluorescence indicating insertion of Badan into the hydrophobic interior of the lipid bilayers. However, the increase in fluorescence was completely dependent on the interaction of Badan-labeled LukF with the gamma-hemolysin second component. Individual spots of Badan fluorescence on erythrocyte membranes were visualized that were associated with single pores. Analyses of the intensities of these fluorescent spots and their photobleaching independently showed that a single pore contained 3-4 LukF molecules. Thus, environment-sensitive fluorophore signals can be used to study the insertion of specific protein domains into cell membranes at the single-molecule lever, and the use of this approach in the present study revealed that a single gamma-hemolysin pore opening contains at least three LukF molecules.
Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan.
Anh Hoa
AH
Nguyen
Vananh T
VT
Nguyen
Yoshiyuki
Y
Kamio
Hideo
H
Higuchi
D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006460:Hemolysin Proteins; D057927:Hydrophobic and Hydrophilic Interactions; D007956:Leukocidins; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000378:metabolism
C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D007956:Leukocidins; C037956:gamma-hemolysin, Staphylococcus aureus; C074300:LukF protein, Staphylococcus aureus; D015081:2-Naphthylamine