Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance...

Area of Science:

• Structural biology
• Biophysics
• Computational biology

Background:

• Protein folding pathways are complex, often involving transient intermediates that are difficult to study.
• The Drosophila melanogaster Engrailed homeodomain (EnHD) is a well-studied model system for protein folding.

Purpose of the Study:

• To structurally characterize the folding intermediates of EnHD using advanced computational and experimental methods.
• To determine the population and role of different conformational states during EnHD folding.

Main Methods:

• Molecular dynamics (MD) simulations were used to generate conformational ensembles for EnHD.
• Small/wide-angle X-ray scattering (SAXS/WAXS) data at four temperatures were used to select relevant structural clusters.
• Nuclear Magnetic Resonance (NMR) spectroscopy with residual dipolar couplings (RDCs) corroborated the structural findings.

Main Results:

• The study identified and characterized native, intermediate, and unfolded states of EnHD.
• A helical intermediate was confirmed and found to be highly populated at the thermal midpoint.
• The fully unfolded state was present at a low fraction across the temperature range.

Conclusions:

• The findings support the proposed folding pathway of EnHD.
• The helical intermediate can be considered the denatured state under physiological conditions.
• Combining ensemble structural techniques with MD enables the determination of structures and populations of interconverting states in solution.