Hydrodynamic properties of colicin A. Existence of a high-affinity lipid-binding site and oligomerization at acid pH.
The hydrodynamic properties of colicin A have been studied. The molecular mass of colicin A was determined from sedimentation equilibrium centrifugation to be 63 +/- 1.2 kDa, in agreement with that determined from the primary amino acid sequence [Morlon et al. (1983) J. Mol. Biol. 110, 271-289]. The sedimentation coefficient has been analyzed over a wide range of ionic strength (NaCl 0.06-0.56 M) and pH (8-4) and was found to remain almost constant. However, below pH 5 an oligomerization of colicin A to tetramers occurred. The frictional coefficient value indicated that the shape of the colicin A monomer was very asymmetric. Analysis of the pH dependence of circular dichroism of colicin A and of its COOH-terminal domain indicated that a sharp transition occurred between pH 4 and 3. This transition was very much reduced for the COOH-terminal domain in the presence of a non-ionic detergent. The presence of a lipid-binding site in colicin A at neutral pH was demonstrated both by hydrodynamic studies with micelles of n-hexadecanoyl and n-octadecanoylphosphocholine and by differential sensitivity to a proteolytic enzyme in the presence or absence of detergent micelles. About 75 molecules of lipid were bound under these conditions suggesting that colicin A was bound to lipid micelles. In contrast, at acid pH, in the presence of an excess of lipid the tetramer was dissociated into monomers complexed to 20-30 lipid molecules, indicating the exposure of a high-affinity lipid-binding site.
Cavard
D
D
Centre de Biochimie et de Biologie Moléculaire du Centre National de la Recherche Scientifique, Marseille, France.
Sauve
P
P
Heitz
F
F
Pattus
F
F
Martinez
C
C
Dijkman
R
R
Lazdunski
C
C
D001419:Bacteria / Q000032:analysis; D001665:Binding Sites; D003087:Colicins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000494:pharmacology
D003087:Colicins; D046911:Macromolecular Substances; D010447:Peptide Hydrolases