Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Nuclear Magnetic Resonance (NMR) spectroscopy is crucial for determining molecular structures.
• Paramagnetic relaxation enhancements (PREs) offer a method for measuring long-range distances in biomolecules, aiding the study of large protein complexes.
• Site-directed labeling is essential for introducing probes at specific locations within proteins.

Purpose of the Study:

• To apply site-directed labeling with paramagnetic probes to the bacterial DnaB helicase.
• To utilize PREs for determining long-range distance restraints in the DnaB helicase.
• To investigate the oligomeric state of the DnaB helicase.

Main Methods:

• Site-directed labeling of the DnaB helicase with maleimide tags carrying nitroxide radicals or lanthanide ions.
• Quantitative continuous-wave electron paramagnetic resonance (EPR) for monitoring labeling success.
• Solid-state NMR spectroscopy (2D and 3D) to extract site-specific PREs.
• Computational modeling to predict PRE values and spin label-nucleus distances.
• Gadolinium (Gd³⁺)-Gadolinium (Gd³⁺) dipolar electron-electron resonance EPR experiments.

Main Results:

• Successful site-directed labeling of the DnaB helicase was achieved.
• Experimental PREs showed good agreement with computationally predicted values.
• The 'blind sphere' size around paramagnetic centers was determined for nitroxide (∼11 Å) and Gd³⁺ (∼14 Å) in ¹³C-detected 2D spectra.
• Gd³⁺-Gd³⁺ EPR experiments confirmed the hexameric assembly of the DnaB helicase.

Conclusions:

• Paramagnetic labeling and PREs are effective for structural studies of large protein complexes like DnaB helicase.
• The study provides insights into the structural organization and assembly of the DnaB helicase.
• The findings contribute to advancing NMR-based structural biology techniques for complex biomolecules.