Figure 2

Oligomerization of SecA-N68 is Mediated by Unstructured Termini (A) The oligomerization of H₆-SecA-N68 and SecA-N68∆NC was characterized by gel filtration chromatography. H₆-SecA-N68 is a SecA construct with an unstructured extreme N-terminal sequence MHHHHHHLTKVFGSRNDRTL (the wild-type sequence is MLIKLLTKVFGSRNDRTL) and also an unstructured C-terminal sequence; SecA-N68∆NC is the same construct, except that the unstructured N- and C-terminal sequences have been removed. H₆-SecA-N68 was applied to the column at concentrations of 4 mg•mL⁻¹ (solid black curve), 2 mg•mL⁻¹ (dashed curve), 0.6 mg•mL⁻¹ (dashed-dotted curve), and 0.35 mg•mL⁻¹ (dotted curve); SecA-N68∆NC (grey curve) was applied at 10 mg•mL⁻¹. The elution volume for molecular weight standards is shown on the upper axis. Panels B to E are SedFit analyses from sedimentation velocity AU of 4 different SecA-N68 constructs at a range of concentrations; all constructs were analyzed under identical buffer conditions consisting of 50 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM EDTA, 5 mM MgCl₂ and 5 mM TCEP-HCl. (B) SecA-N68, with a wild-type N-terminal sequence, was analyzed at concentrations of 1.0 (dashed) and 5.0 (solid) mg•mL⁻¹. (C) SecA-N68ΔNC, which lacks both unstructured termini, was analyzed at concentrations of 0.1 (dashed) and 4 (solid) mg•mL⁻¹. (D) Analysis of SecA-N68ΔC, which carries only the wild-type unstructured N-terminus, at concentrations of 0.25 (dotted), 1 (dashed), and 2 (solid) mg•mL⁻¹. (E) Analysis of SecA-N68ΔN, which carries only the unstructured C-terminus, at concentrations of 0.4 (dotted), 0.6 (dashed), and 4 (solid) mg•mL⁻¹.