Single-Molecule Visualization of Environment-Sensitive Fluorophores Inserted into Cell Membranes by Staphylococcal γ-Hemolysin

• Published in: Biochemistry (Easton) Vol. 45; no. 8; pp. 2570 - 2576
• Main Authors: Nguyen, Anh Hoa, Nguyen, Vananh T, Kamio, Yoshiyuki, Higuchi, Hideo
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 28-02-2006
• Subjects: 2-Naphthylamine - analogs & derivatives; 2-Naphthylamine - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Proteins - pharmacology; Bacterial Toxins - metabolism; Bacterial Toxins - pharmacology; Cell Membrane - metabolism; Erythrocytes - metabolism; Fluorescent Dyes - metabolism; Hemolysin Proteins; Hydrophobic and Hydrophilic Interactions; Leukocidins; Microscopy, Fluorescence; Models, Biological; Protein Structure, Tertiary; Staphylococcus aureus - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0514156

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 γ-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 γ-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 γ-hemolysin pore opening contains at least three LukF molecules.