Poster Presentation 2014 International Biophysics Congress

A method to gain insight into the functioning of Colicin A (#307)

Ana Bouchet 1 2 , Ane Metola 1 , Alain Ibañez de Opakua 1 3 , Anibal Disalvo 2 , Félix Goñi 1 4 , Ana Rosa Viguera Rincón 1
  1. Unidad de Biofisica, CSIC-UPV/EHU, Bilbao, Spain
  2. CITSE-CONICET, Santiago Del Estero, Argentina
  3. Structural Biology Unit, CIC bioGUNE, Bilbao, Spain
  4. Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain
Colicin A is a bacteriocin released by E. coli under stress conditions. It enters into the cell through the Tol machinery that drives the C-terminal domain (pf-ColA) to the inner membrane to depolarize and kill the cell (1). Extensive in vitro functional studies carried out on liposomes and black lipid films have contributed to demonstrate its pore forming capacity. The activity assigned to pore forming colicins ranges from ion conductance (2) to extensive leakage (3) and even membrane fusion (4). The diversity of protocols used to reconstitute the protein and different solvent and lipidic environments employed might explain the discrepancies found in the literature.In this work we reconstituted native and non-native pf-ColA into liposomes of several lipid compositions including those mimicking the native lipid environment. We found that the in vitro conditions of pf-ColA unfolding are critical to achieve a functional insertion of the protein into the lipid membrane. Increasing incorporated protein relative to lipid allowed to distinguish activities that ranged from (i) proton conductance, (ii) cation passage, (iii) leakage to (iv) liposome aggregation. Experiments carried out with liposomes made of E. coli total lipids extract, at near neutral pH and very high lipid:protein ratios, similar to those feasible in physiological scenarios, are compatible with selective proton permeability facilitated by pf-ColA monomers.