Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state...

Area of Science:

• Biophysics
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Determining high-resolution protein structures is crucial for understanding biological function.
• Solid-state NMR spectroscopy offers a powerful tool for studying proteins in their native-like states.
• Previous methods had limitations in obtaining precise internuclear distance restraints for complete structure determination.

Purpose of the Study:

• To demonstrate the capability of magic-angle spinning solid-state NMR spectroscopy for determining complete, high-resolution protein structures.
• To establish a method for obtaining quantitative internuclear distances in proteins.
• To validate the precision and accuracy of the obtained structural data.

Main Methods:

• Utilized three-dimensional ZF-TEDOR pulse sequence with sparse labeling of carbon-13 (13C) sites in the beta1 domain of immunoglobulin binding protein G (GB1).
• Employed bacterial expression with specific 13C-labeled glycerol sources for protein labeling.
• Extracted quantitative dipolar trajectories from two-dimensional nitrogen-15 (15N)-13C planes and fitted data to theoretical spin cluster trajectories.

Main Results:

• Resolved approximately 750 cross peaks in 2D (15)N-(13)C planes, enabling quantitative distance measurements.
• Achieved high precision (up to 0.1 Å) for approximately 350 internuclear distances, with good precision for others in the 5-8 Å range.
• Generated a highly precise protein structure (backbone RMSD of 0.25±0.09 Å) using distance and dihedral angle restraints, showing excellent agreement with crystal structures.

Conclusions:

• Quantitative internuclear distances can be reliably measured in solid proteins using magic-angle spinning solid-state NMR.
• This technique enables the determination of complete, atomic-resolution structures of moderately sized proteins.
• The study validates solid-state NMR as a method for high-precision structural biology.