Investigation of Conformation Changes of Bothropstoxin-I (PLA2-Lys49) from the venom of Bothrops jararacussu on Association with Artificial Membranes. 

Ferreira T. L.³, Ruller R.², Chioato L.³, Ward R. J.¹

1 Depto. de Química da FFCLRP, 2 Depto. de Biologia Molecular e Celular e Bioagentes Patogênicos, 3 Depto. Bioquímica e Imunologia FMRP.

Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-PLA2 present in the venom of the snake Bothropsjararacussu. Although lacking hydrolytic activity against phospholipid substrates, BthTx-I damages artificial membranes by a Ca²⁺-independent mechanism. We have previously suggested that a transition between “open” and “closed” membrane bound dimer conformations is responsible for this activity. In order to study the interaction of the BthTx-I with model membranes by intrinsic tryptophan (Trp) fluorescence spectroscopy, the single Trp77 residue at the dimer interface was substituted by histidine (W77H), and in a second round of mutagenesis 10 single Trp mutants were produced to create double mutants with single Trp residues located in the dimer interface, interface recognition site and C-terminal loop regions. Liposome membrane damage by the C-terminal loop mutants (at positions 117,119 and 125) was reduced in the case of the W77H/F125W mutant. Trp emission spectra from mutants W77H/Y117W and W77H/F125W revealed changes in the presence of liposomes suggesting that the microenvironments of these residues is altered in the membrane bound form of the protein. These results support the hypothesis that the C-terminal loop region maintains an intimate contact with the target membrane, however does not provide evidence in favour of insertion of this region into the membrane bilayer.

Support; FAPESP, CNPq and PRP-USP and FAEPA

Correspondence to: tathylf@hotmail.com