Homodimeric Escherichia coli Toxin CcdB (Controller of Cell Division or Death B Protein) Folds via Parallel Pathways

Abstract

The existence of parallel pathways in the folding of proteins seems intuitive, yet remains controversial. We explore the folding kinetics of the homodimeric Escherichia coli toxin CcdB (Controller of Cell Division or Death B protein) using multiple optical probes and approaches. Kinetic studies performed as a function of protein and denaturant concentrations demonstrate that the folding of CcdB is a four-state process. The two intermediates populated during folding are present on parallel pathways. Both form by rapid association of the monomers in a diffusion limited manner and appear to be largely unstructured, as they are silent to the optical probes employed in the current study. The existence of parallel pathways is supported by the insensitivity of the amplitudes of the refolding kinetic phases to the different probes used in the study. More importantly, interrupted refolding studies and ligand binding studies clearly demonstrate that the native state forms in a biexponential manner, implying the presence of at least two pathways. Our studies indicate that the CcdA antitoxin binds only to the folded CcdB dimer and not to any earlier folding intermediates. Thus, despite being part of the same operon, the antitoxin does not appear to modulate the folding pathway of the toxin encoded by the downstream cistron. This study highlights the utility of ligand binding in distinguishing between sequential and parallel pathways in protein folding studies, while also providing insights into molecular interactions during folding in Type II toxin-antitoxin systems.

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

“…Refolding and unfolding kinetics following manual mixing for CcdB was monitored by time‐course fluorescence spectroscopy as a function of time at 25 °C. Refolding was carried out with 2 μM of purified proteins in either 10 or 200 mM HEPES, at pH 8.4 at a final GdnCl of 1.5 M. Refolding for CcdB occurs with a significant burst phase and a slow phase as observed earlier with measured rates similar in both the instruments in both buffer conditions (Fig. , Table ).…”

“…Refolding and unfolding kinetics for CcdB were monitored by both fluorimeter (fluorescence intensity at 340 nm) and nanoDSF (F 350 /F 330 using PR. TimeControl software, Prometheus NT.48) at 25 °C.…”

“…Unfolding kinetic traces of fluorescence intensity in 3 M GdnCl as a function of time for CcdB in 10 mM or 200 mM HEPES, pH 8.4 were normalized from 0 to 1 between native and denatured baseline at 3 M GdnCl. The data was analyzed using SigmaPlot™ version 12.5 for Windows™ scientific graphing software, from Systat Software, Inc., San Jose, CA, USA, (http://www.systatsoftware.com), and plots were fitted to a 5 parameter equation for exponential decay for refolding (y = y 0 + a*exp(−bx) + c*exp(−dx)), yielding slow and fast phase rate constants and a 3 parameter exponential rise for unfolding (y = y 0 + a*exp[b*x]) as described previously, where x is the time (in seconds) of refolding/unfolding …”

“…Protein stability is either thermodynamically or kinetically controlled . Widely used techniques for deciphering thermodynamic and kinetic stability of proteins involve measurements of intrinsic fluorescence either at equilibrium or as a function of time . Such measurements are sample and labor intensive.…”

“…We describe equilibrium, isothermal, unfolding studies for two proteins, homodimeric Escherichia coli CcdB and monomeric hen egg white lysozyme (HEWL) using both a conventional fluorimeter and nanoDSF. We also measure unfolding and refolding kinetics of CcdB using nanoDSF and compare with previously published kinetic data . The data demonstrate that nanoDSF can be used to accurately and rapidly measure protein stability and folding/unfolding kinetics with minimal sample consumption.…”