Structure-function studies of tryptophan mutants of equinatoxin II, a sea anemone pore-forming protein

被引:79
作者
Malovrh, P
Barlic, A
Podlesek, Z
Macek, P
Menestrina, G
Anderluh, G
机构
[1] Univ Ljubljana, Dept Biol, Biotech Fac, Ljubljana 1000, Slovenia
[2] CNR, ITC, Ctr Fis Stati Aggregati, I-38050 Povo, Trento, Italy
关键词
actinoporin; Actinia equina; lipid membranes; fluorescence; FTIR spectroscopy;
D O I
10.1042/0264-6021:3460223
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Equinatoxin II (EqtII) is a eukaryotic cytolytic toxin that avidly creates pores in natural and model lipid membranes. It contains five tryptophan residues in three different regions of the molecule, In order to study its interaction with the lipid membranes, three tryptophan mutants, EqtII Trp(45), EqtII Trp(116/117) and EqtII Trp(149), were prepared in an Escherichia coli expression system [here, the tryptophan mutants are classified according to the position of the remaining tryptophan residue(s) in each mutated protein]. They all possess a single intrinsic fluorescent centre. All mutants were less haemolytically active than the wild-type, although the mechanism of erythrocyte damage was the same. EqtII Trp(116/117) resembles the wild-type in terms of its secondary structure content, as determined from Fourier-transform infrared (FTIR) spectra and its fluorescent properties. Tryptophans at these two positions are buried within the hydrophobic interior of the protein, and are transferred to the lipid phase during the interaction with the lipid membrane. The secondary structure of the other two mutants, EqtII Trp(45) and EqtII Trp(149), was altered to a certain extent. EqtII Trp(149) was the most dissimilar from the wild-type, displaying a higher content of random-coil structure. It also retained the lowest number of nitrogen-bound protons after exchange with (H2O)-H-2, which might indicate a reduced compactness of the molecule. Tryptophans in EqtII Trp(45) and EqtII Trp(149) were more exposed to water, and also remained as such in the membrane-bound form.
引用
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页码:223 / 232
页数:10
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