Isolation, characterization and lipid-binding properties of the recalcitrant FtsA division protein from Escherichia coli

Abstract

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures.

Figure 1. Molecular characterization of purified E. coli FtsA protein.

(A) SDS-PAGE analysis. Loaded FtsA concentration was 7 µM. Molecular weight markers are in the left lane. (B) Far-UV circular dichroism spectra (CD) of denatured (dashed) and refolded FtsA (solid). (C) Corrected fluorescence emission spectra of denatured (dashed) and refolded (solid) FtsA. λ_exc = 295 nm, 20°C. (D) Thermal unfolding of FtsA as monitored by CD.

Figure 2. Sedimentation coefficient c(s) distribution of E. coli FtsA (10 µM) in working buffer with 0.1 mM ADP and 0.2 mM TCEP, generated from the sedimentation velocity interference data.

Interaction of E. coli FtsA with lipid/membrane structures.

(A) FtsA binding to large unilamellar liposomes made of E. coli phospholipids (solid) or inner membrane vesicles (dashed) as monitored by turbidity. (B) FtsA binding to micro-beads coated with E. coli lipids (circles), inner membrane (triangles) and phosphatidylcholine (squares). Data points correspond to the average of at least three individual measurements ± SD. Solid lines represent the best fit of equation (1) to the data with the best-fit parameters shown in the figure. (C) Effect of ionic strength on FtsA binding to EcPL-beads. 100 (circles), 300 (triangles) and 500 mM KCl (diamonds). Errors bars are omitted for the sake of clarity. Dashed line is just intended to guide the eye.