Complex behavior in solution of homodimeric SecA

Abstract

SecA, a homodimeric protein involved in protein export in Escherichia coli, exists in the cell both associated with the membrane translocation apparatus and free in the cytosol. SecA is a multifunctional protein involved in protein localization and regulation of its own expression. To carry out these functions, SecA interacts with a variety of proteins, phospholipids, nucleotides, and nucleic acid and shows two enzymic activities. It is an ATPase and a helicase. Its role during protein localization involves interaction with the precursor polypeptides to be exported, the cytosolic chaperone SecB, and the SecY subunit of the membrane-associated translocase, as well as with acidic phospholipids. At the membrane, SecA undergoes a cycle of binding and hydrolysis of ATP coupled to conformational changes that result in translocation of precursors through the cytoplasmic membrane. The helicase activity of SecA and its affinity for its mRNA are involved in regulation of its own expression. SecA has been reported to exist in at least two conformational states during its functional cycle. Here we have used analytical centrifugation, as well as column chromatography coupled with multi-angle light scatter, to show that in solution SecA undergoes at least two monomer-dimer equilibrium reactions that are sensitive to temperature and to concentration of salt.

Fig. 1.

Size-exclusion chromatography of SecA at various concentrations. Two-hundred μL samples of pure SecA were subjected to size-exclusion chromatography at 8°C. The concentration of SecA in samples loaded on the column were 1, 2, 4, 8, 12, and 20 μM (expressed as monomer). The peak of absorbance at 280 nm eluted at ∼18.3 mL for the 1 μM sample. The peak eluted earlier as the sample concentration was increased. Absorbance at 280 nm was normalized to the maximum peak height of the individual runs for comparison.

Fig. 2.

Sedimentation velocity centrifugation of SecA at 6°C. (A) The analytical ultracentrifuge cell contained 10 μM SecA monomer in 10 mM HEPES, 300 mM K⁺ acetate, 5 mM Mg⁺² acetate, 1 mM TCEP, pH 7.5. Twenty-eight successive scans are shown. Scans 3–28 were subjected to analysis as shown in panel B. The middle 90% of the boundary was divided into 50 sections as described in the text. The midpoint of the boundary is indicated by the arrow. (B) The raw data shown in panel A were subjected to analysis by the method of Van Holde and Weischet (1978). Each point is the apparent sedimentation coefficient (s*) of each fraction of the boundary, and each vertical array of 50 points represents one boundary. Each line is the best fit through the points for a particular boundary fraction. The arrow indicates boundary fraction 0.5. (C) SecA was subjected to centrifugation, and the data were analyzed as described above. The intercepts of each line in the van Holde-Weischet analysis were plotted vs. the boundary fraction to which the line pertained. The concentrations of SecA monomer subjected to centrifugation were 1.6 μM (▪), 6 μM (•), 10 μM (▴), and 24 μM (○).

Fig. 3.

Size-exclusion chromatography of SecA at 24°C. Two-hundred μL samples of pure SecA were subjected to size-exclusion chromatography at 24°C. The concentrations of SecA monomer of the samples loaded on the column were 4 μM (dotted line), 8 μM (dashed line), and 12 μM (solid line). Absorbance at 280 nm was normalized to the maximum height of the SecA peak eluting earliest.

Fig. 4.

Temperature dependence of SecA equilibrium reactions. SecA was applied at a concentration of 8 μM (monomer) and chromatography performed at 8°C and at 24°C. Oxidized SecA was applied at 5 μM (i.e., 2.5 μM dimer) and chromotographed at 8°C. Molar mass was determined as described in text. The lines represent relative absorbance at 280 nM. The symbols for molar mass are aligned with the peaks from which the values were derived as follows: Oxidized SecA (♦), SecA at 8°C (○), and SecA at 20°C (▪).

Fig. 5.

Sedimentation velocity centrifugation of SecA. Analyses were performed as described in the legend of Figure 2 ▶. SecA at 6 μM monomer was subjected to centrifugation at 6°C (♦) and at 20°C (•). Oxidized SecA, which is a covalent dimer, was centrifuged at 5 μM (expressed as monomer) at both 6°C (▪) and 20°C (▴). The backward curvature of the distribution at 20°C is indicative of non-ideal behavior, which might result from one of a number of causes including asymmetric shape of the particle or high charge density.

Fig. 6.

Molar mass of SecA as it elutes from the size-exclusion column. A 200-μL sample of SecA, 12 μM monomer, was subjected to size-exclusion chromatography at 8°C. As the protein eluted from the column, absorbance at 280 nm was monitored (solid line) and the molar mass determined by light scatter (dots).

Fig. 7.

Estimation of apparent equilibrium constant. The weight average molar mass of the top 1% of the absorbance peak at 280 nm of SecA eluted from a size-exclusion column was determined as described. Circles represent wild-type SecA from three different protein preparations, and inverted triangles represent two preparations of SecAN880, a truncated form of SecA. Ideal curves are shown for a monomer-dimer equilibrium for which the monomer molar mass is 102 kD and the equilibrium constant expressed for the dissociation reaction is 0.5 μM (dotted line), 1.0 μM (solid line), 1.5 μM (dashed lined), and 2.0 μM (dotted-dashed line).