Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.
The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.
Biomembrane Laboratory, Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India.
S M
SM
Raja
S S
SS
Rawat
A
A
Chattopadhyay
A K
AK
Lala
D001427:Bacterial Toxins / Q000737:chemistry* / Q000633:toxicity*; D055592:Biophysical Phenomena; D001703:Biophysics; D001974:Bromosuccinimide; D003497:Cyclic N-Oxides; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000633:toxicity*; D007454:Iodides; D008566:Membranes / Q000737:chemistry; D010713:Phosphatidylcholines; D011487:Protein Conformation; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000472:pathogenicity*; D013229:Stearic Acids; D014364:Tryptophan / Q000737:chemistry; D014867:Water
D001427:Bacterial Toxins; D003497:Cyclic N-Oxides; D006460:Hemolysin Proteins; D007454:Iodides; D010713:Phosphatidylcholines; D013113:Spin Labels; D013229:Stearic Acids; C021091:staphylococcal alpha-toxin; D014867:Water; C060803:9,10-dibromostearic acid; C049875:1,2-di(9,10-dibromostearoyl)phosphatidylcholine; D014364:Tryptophan; D001974:Bromosuccinimide