Simultaneous NMR study of protein structure and dynamics using conservative mutagenesis

Area of Science:

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

Background:

• Determining internuclear bond vector orientation and dynamics is crucial for understanding protein structure and function.
• Nuclear Magnetic Resonance (NMR) dipolar couplings provide valuable information about molecular structure and dynamics.
• Previous methods often require multiple experimental conditions or complex data analysis.

Purpose of the Study:

• To develop a novel iterative procedure for determining orientation and dynamics of internuclear bond vectors from NMR dipolar couplings.
• To assess the degree of motional anisotropy and the direction of largest amplitude internal motion.
• To validate the method using a well-studied protein domain (GB3) and compare results with existing techniques.

Main Methods:

• Utilizing NMR dipolar couplings measured under at least three orthogonal alignment conditions.
• Employing conservative mutations of charged surface residues to achieve alignment variation.
• Applying an iterative procedure for direct interpretation of the NMR data.
• Analyzing backbone (15)N-(1)H, (13)C(alpha)-(1)H(alpha), and (13)C(alpha)-13C' interactions in GB3 protein domain.

Main Results:

• The method successfully determined orientation and dynamics of backbone bond vectors in GB3.
• Results showed remarkably uniform backbone dynamics for residues in secondary structures, consistent with (15)N relaxation studies.
• Residues in loop and turn regions exhibited larger amplitude dynamics than previously reported.
• Elevated dynamics were observed for N-H bonds in the second beta-strand, involved in antibody recognition, with motion orthogonal to the chain direction.

Conclusions:

• The novel iterative NMR procedure provides a robust method for characterizing protein backbone dynamics and orientation.
• The findings highlight variations in protein dynamics between secondary structure elements and loop/turn regions.
• The method's ability to detect localized dynamic behaviors, such as in antibody recognition sites, is significant for structural biology.