Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A.
The intrinsic fluorescence of the colicin A thermolytic fragment does not change after insertion into normal phospholipid vesicles and is thus an unsuitable probe for monitoring the membrane insertion process. In this paper, we report the results of studies on the quenching of this fluorescence by brominated dioleoylphosphatidylglycerol (Br-DOPG) vesicles. Bromine atoms located at the midpoint of the phospholipid acyl chain quench the tryptophan fluorescence, indicating contact between fluorophores of the protein and the bilayer's hydrophobic core. Addition of Br-DOPG vesicles to a protein solution quenches the tryptophan fluorescence in a time-dependent manner. This quenching can be fitted to a single-exponential function, and thus interpreted as a one-step process. This allows calculation of an apparent rate constant of protein insertion into the membrane. Parameters known to affect the insertion of the thermolytic fragment into phospholipid monolayers or vesicles (pH and negative charge density) also affect the rate constant in comparable ways. In addition to the information gained concerning membrane exposure in the steady state, this approach provides the first real-time method for measuring the insertion of colicin into membranes. It is highly quantitative and can be used on all versions of the protein, e.g., full size, proteolytic fragments, and mutants. Brominated lipids provide experimental conditions identical to normal lipids and allow for great flexibility in protein/lipid ratios and concentrations. The kinetic analysis shows clearly the existence of a two-step process involving a rapid adsorption of the protein to the lipid surface followed by a slow insertion.
González-Mañas
J M
JM
European Molecular Biology Laboratory, Heidelberg, Germany.
Lakey
J H
JH
Pattus
F
F
D016971:Citrobacter freundii / Q000737:chemistry / Q000254:growth & development; D003087:Colicins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers; D008961:Models, Structural; D010713:Phosphatidylcholines / Q000138:chemical synthesis / Q000737:chemistry*; D010715:Phosphatidylglycerols / Q000138:chemical synthesis / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013820:Thermolysin
D003087:Colicins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D013820:Thermolysin