Double Electron-Electron Spin Resonance Tracks Flavodoxin Folding

Area of Science:

• Biochemistry and Molecular Biophysics
• Structural Biology
• Biophysical Chemistry

Background:

• Protein folding is a fundamental challenge in biochemistry, crucial for understanding protein function.
• Mapping protein structure during folding is essential for elucidating folding pathways.
• Pulsed electron paramagnetic resonance (EPR) spectroscopy offers potential for distance measurements in biomolecules.

Purpose of the Study:

• To evaluate the utility of distance determination between paramagnetic spin-labels using pulsed EPR.
• To investigate the denaturant-dependent equilibrium folding of flavodoxin using double electron-electron spin resonance (DEER).
• To characterize conformational changes during protein unfolding.

Main Methods:

• Utilized double electron-electron spin resonance (DEER), a pulsed EPR technique, for distance measurements.
• Employed spin-labeling with MTSL ((1-oxy-,2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate) at positions 69 and 131 of flavodoxin.
• Studied the equilibrium folding of flavodoxin across a range of guanidine hydrochloride concentrations.

Main Results:

• Nativelike spin-label separation was observed up to 0.8 M guanidine hydrochloride, indicating a stable native state.
• An additional distance distribution component emerged at 2.3 M denaturant, attributed to a folding intermediate.
• Increased denaturant concentrations led to protein expansion and a broader range of conformations compared to the native state.

Conclusions:

• DEER spectroscopy is a powerful and versatile technique for studying protein folding dynamics.
• The study successfully mapped conformational changes during flavodoxin unfolding, identifying intermediate states.
• DEER expands the methodological toolkit for investigating complex protein folding processes.