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ISSN 1027-2852 (Electronic)
ISSN 0864-4551 (Print)

2011, Number 1

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Biotecnol Apl 2011; 28 (1)

The Sticholysin I mutants St I E2C and St I R52C show similar binding to liposomal vesicles but differ in their permeabilizing activity

López A, Valle A, Pedrera L, Martínez D, Fando R, Schreier S, Álvarez C, Lanio ME, Pazos F

Language: English
References: 38
Page: 13-18
PDF size: 211.76 Kb.


ABSTRACT

The mechanism of pore formation by actinoporins is a multistep process, involving binding of water soluble monomer to membrane and subsequent oligomerization of monomers on the membrane surface, forming a functional pore. However, molecular details of membrane insertion mechanism and oligomerization are not clear. A phosphocholine-binding site and a surface cluster of aromatic rings, together with a basic region, are important to the initial interaction with membrane and the N-terminal region is relevant in the pore formation. Aiming to deepen into the structure-function relationship in sticholysins, we designed and produced two Cys mutants of recombinant sticholysin I (rSt I) in relevant functional regions for membrane interaction: St I E2C (in the N-terminal region) and St I R52C (in the membrane binding site). Conformational studies suggested that the replacement of Glu-2 and Arg-52 by a Cys residue in rSt I not noticeably changes protein conformation as assessed by fluorescence and CD spectroscopy, the first change not affecting toxin’s permeabilizing ability. The relative decrease in the pore forming capacity of St I R52C is not related with a smaller binding capacity of this mutant to membrane. In summary, St I E2C and St I R52C retain the main conformational properties of the wild type and show similar binding to liposomal vesicles while differing in their permeabilizing activity. St I E2C and St I R52C constitute good tools to study those steps of the permeabilizing mechanism of sticholysins that take place after binding to membrane, using thiol-specific probes such as fluorescent and spin labels.


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