Topography studies on the membrane interaction mechanism of the eosinophil cationic protein.
The eosinophil cationic protein (ECP) is an antipathogen protein involved in the host defense system. ECP displays bactericidal and membrane lytic capacities [Carreras et al. (2003) Biochemistry 42, 6636-6644]. We have now characterized in detail the protein-membrane interaction process. All observed fluorescent parameters of the wild type and single-tryptophan-containing mutants, as well as the results of decomposition analysis of protein fluorescence, suggest that W10 and W35 belong to two distinct spectral classes I and III, respectively. Tryptophan residues were classified and assigned to distinct structural classes using statistical approaches based on the analysis of tryptophan microenvironment structural properties. W10 belongs to class I and is buried in a relative nonpolar, nonflexible protein environment, while W35 (class III) is fully exposed to free water molecules. Tryptophan solvent exposure and the depth of the protein insertion in the lipid bilayer were monitored by the degree of protein fluorescence quenching by KI and brominated phospholipids, respectively. Results indicate that W35 partially inserts into the lipid bilayer, whereas W10 does not. Further analysis by electron microscopy and dynamic light scattering indicates that ECP can destabilize and trigger lipid vesicle aggregation at a nanomolar concentration range, corresponding to about 1:1000 protein/lipid ratio. No significant leakage of the vesicle aqueous content takes place below that protein concentration threshold. The data are consistent with a membrane destabilization "carpet-like" mechanism.
Torrent
Marc
M
Departamento de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autonoma de Barcelona, 08193-Bellaterra, Spain.
Cuyás
Elisabet
E
Carreras
Esther
E
Navarro
Susanna
S
López
Olga
O
de la Maza
Alfons
A
Nogués
M Victòria
MV
Reshetnyak
Yana K
YK
Boix
Ester
E
D047150:Eosinophil Cationic Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005614:Freeze Fracturing; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008854:Microscopy, Electron; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D008051:Lipid Bilayers; D008081:Liposomes; D014364:Tryptophan; D047150:Eosinophil Cationic Protein