Side chain dynamics monitored by 13C-13C cross-relaxation.

Area of Science:

• Biophysics
• Structural Biology
• Protein Dynamics

Background:

• Understanding protein side chain mobility is crucial for deciphering protein function.
• Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful tool for studying protein structure and dynamics.

Purpose of the Study:

• To develop and validate a novel method for measuring carbon-carbon (¹³C-¹³C) cross-relaxation rates in uniformly ¹³C-labeled proteins.
• To apply this method to investigate the mobility of side chains in the serine protease PB92.

Main Methods:

• Utilized a modified (H)CCH-NOESY pulse sequence with zero-quantum (ZQ) coherence suppression.
• Extracted initial cross-relaxation rates from Nuclear Overhauser Effect (NOE) buildup curves.
• Applied the method to a 269-residue serine protease (PB92).

Main Results:

• Successfully measured C(alpha)-C(beta) cross-relaxation rates for 64% of residues and methyl ¹³C-¹³C cross-relaxation rates for 47% of methyl-containing C-C pairs.
• Observed higher C(alpha)-C(beta) cross-relaxation rates in secondary structures compared to loop regions, indicating greater rigidity.
• Demonstrated a decrease in methyl ¹³C-¹³C cross-relaxation rates further from the main chain, signifying increased flexibility.

Conclusions:

• The developed method provides valuable insights into protein side chain dynamics.
• Protein secondary structures exhibit higher rigidity than loop regions.
• Side chain flexibility increases with distance from the protein backbone, with anisotropic motion observed in leucine residues.