Switching Protein Conformational Substates by Protonation and Mutation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein modules regulate cellular responses by controlling transcription factor availability and conformation.
• Cnu, a four-helix bundle protein, functions as a thermosensor for virulence gene expression in enterobacteriaceae.
• Cnu is also implicated in pH-dependent regulation of virulence genes.

Purpose of the Study:

• To investigate the mechanism by which Cnu senses and responds to pH changes.
• To determine the role of a conserved buried histidine (H45) in Cnu's pH-sensing ability.
• To elucidate how pH-induced conformational changes affect Cnu's interaction with transcription factors like H-NS.

Main Methods:

• Spectroscopic and calorimetric techniques were employed to study Cnu's conformational changes.
• A Wako-Saitô-Muñoz-Eaton (WSME) statistical mechanical model was used for computational predictions.
• Fluorescence lifetime measurements of a tryptophan residue validated computational predictions.

Main Results:

• Cnu's histidine residue (H45) has a suppressed pKa of ~5.1, and its protonation triggers a conformational switch.
• Protonation leads to the disordering of Cnu's fourth helix, altering its structure.
• A mutation (H45V) abolished pH-dependent switching, confirming H45's critical role.
• WSME model predictions regarding helix conformations and fluctuations were experimentally validated.

Conclusions:

• Cnu functions as a dual thermo- and pH-sensor.
• The pH-sensing mechanism involves protonation of H45, inducing electrostatic frustration and conformational changes.
• This process is governed by the interplay between structural element packing and histidine protonation.