Protein Structural Ensembles Visualized by Solvent Paramagnetic Relaxation Enhancement

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Proteins exist in multiple conformations to perform their functions, but characterizing these structural ensembles is challenging.
• Established Nuclear Magnetic Resonance (NMR) techniques like paramagnetic relaxation enhancement (PRE) are valuable but often limited to rigid protein structures.

Purpose of the Study:

• To develop and validate a method for characterizing protein dynamics on the microsecond-millisecond timescale.
• To expand the application of PRE NMR to study flexible proteins and identify alternative conformations.

Main Methods:

• Accurate measurement of solvent paramagnetic relaxation enhancement (sPRE) in the presence of an inert paramagnetic cosolute.
• Analysis of sPRE data to assess protein dynamics and structural ensembles.
• Integration with molecular dynamics simulations to identify and validate alternative protein conformations.

Main Results:

• The developed sPRE method successfully characterizes microsecond-millisecond dynamics in multi-domain proteins.
• The analysis of sPRE data reveals ensembles of structures that accurately explain experimental observations.
• The method identified previously theorized alternative protein conformations when combined with molecular dynamics simulations.

Conclusions:

• The novel sPRE-based method expands the utility of PRE NMR beyond rigid proteins.
• This approach provides a powerful tool for investigating protein dynamics and conformational heterogeneity.
• The findings offer new insights into the functional relevance of protein structural ensembles.