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Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus

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Title Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus
 
Creator RAJA, SM
RAWAT, SS
CHATTOPADHYAY, A
LALA, AK
 
Subject alpha-toxin
crystallographic refinement
brominated phospholipids
labeled phospholipids
protein fluorescence
secondary structure
model membranes
colicin-a
channel
hemolysin
 
Description 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 Angstrom 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.
 
Publisher BIOPHYSICAL SOCIETY
 
Date 2011-07-19T01:11:43Z
2011-12-26T12:50:55Z
2011-12-27T05:37:08Z
2011-07-19T01:11:43Z
2011-12-26T12:50:55Z
2011-12-27T05:37:08Z
1999
 
Type Article
 
Identifier BIOPHYSICAL JOURNAL, 76(3), 1469-1479
0006-3495
http://dspace.library.iitb.ac.in/xmlui/handle/10054/5127
http://hdl.handle.net/10054/5127
 
Language en