NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Proteins exhibit a trade-off between stability and bioactivity, shaped by evolutionary pressures.
• Identifying residues that modulate this balance is crucial for understanding protein function.

Purpose of the Study:

• To identify specific amino acid residues in G-CSF that are pivotal to the stability-bioactivity trade-off.
• To investigate the relationship between protein structure, dynamics, and function.

Main Methods:

• Utilized 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy.
• Applied thermal perturbation to probe residue microenvironment and dynamics.
• Integrated NMR data with structure relaxation methods.

Main Results:

• Identified four residues (G4, A6, T133, Q134) critical for global protein stability.
• Observed localized conformational changes in four structural clusters, particularly around loop AB, prior to global unfolding.
• Highlighted residues H43, V48, and S63 as key to loop AB opening motion, potentially linked to function and aggregation.

Conclusions:

• Developed an approach to profile residues involved in protein stability and bioactivity.
• The findings provide insights into G-CSF's conformational dynamics, function, and aggregation propensity.
• This methodology can be applied to other proteins to understand structure-function relationships.