Two-dimensional IR correlation spectroscopy: sequential events in the unfolding process of the lambda cro-V55C...

Area of Science:

• Protein dynamics and biophysics
• Spectroscopic analysis of molecular interactions

Background:

• Protein folding/unfolding mechanisms are debated, with questions about cooperative versus sequential processes.
• Understanding these mechanisms requires advanced analytical tools capable of resolving distinct events.

Purpose of the Study:

• To investigate the sequential nature of protein unfolding using two-dimensional infrared (2D IR) correlation spectroscopy.
• To analyze the thermal unfolding pathway of the Cro-V55C dimer of the lambda Cro repressor.

Main Methods:

• Utilized two-dimensional (2D) IR correlation spectroscopy to analyze IR spectra.
• Recorded spectra over a temperature range of 20-95 degrees C.
• Applied 2D IR correlation analysis to identify sequential events in protein unfolding.

Main Results:

• The thermal unfolding of the Cro-V55C dimer proceeds through a stable equilibrium intermediate.
• Evidence for a sequential formation of the intermediate was observed, occurring in three distinct steps.
• The unfolding involves initial N-terminal beta-strand unfolding, followed by alpha-helix unfolding, and subsequent reorganization of beta-sheets and unordered segments.

Conclusions:

• The unfolding process of the lambda Cro repressor dimer is not fully cooperative but involves sequential events.
• 2D IR correlation spectroscopy is a powerful tool for dissecting complex protein unfolding pathways.
• The stable intermediate undergoes sequential unfolding, culminating in the disruption of dimer interface hydrogen bonds.