CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core

Area of Science:

• Biochemistry
• Structural Biology
• Protein Chemistry

Background:

• Protein structure, stability, and activity are significantly influenced by pH.
• The controller of cell death B (CcdB) protein is a key factor in bacterial cell death regulation.

Purpose of the Study:

• To investigate the structural and stability changes of the homodimeric CcdB protein at low pH (pH 4).
• To understand the equilibrium state of CcdB under acidic conditions and its implications for protein function.

Main Methods:

• Circular dichroism spectroscopy
• 8-anilino-1-naphthalene-sulphonate binding assays
• Trp solvation studies
• Size-exclusion chromatography coupled to multiangle light scattering (SEC-MALS)
• Analytical ultracentrifugation (AUC)
• Electron paramagnetic resonance (EPR)
• 2D ¹⁵N-¹H heteronuclear single-quantum coherence (HSQC) NMR
• Hydrogen-exchange mass spectrometry (HX-MS)

Main Results:

• At pH 4, CcdB adopts a partially unfolded state with a dry core.
• CcdB remains dimeric at pH 4, confirmed by multiple biophysical techniques.
• NMR analysis indicates structural similarity but non-identity between pH 4 and pH 7 states.
• HX-MS reveals anisotropic changes in local stability, with core regions near the dimer interface showing reduced protection.

Conclusions:

• Low pH induces significant structural alterations in CcdB while preserving its dimeric quaternary structure.
• The observed changes in stability and structure at low pH provide insights into CcdB's dynamic behavior and potential functional regulation.