Abstract
FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined α-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1–10 μM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 °C of 15.2kcalmol−1 in urea and 13.5kcalmol−1 in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6kcalmol−1 in urea and 10.5kcalmol−1 in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, α-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, α-helical dimers.
Published Version
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