Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding...

Area of Science:

• Biophysics
• Structural Biology
• Computational Biology

Background:

• Protein dynamics involve transitions between native and transient excited states.
• Excited states are crucial for protein folding and function but difficult to experimentally characterize.
• Flavodoxin fold protein CheY is extensively studied and serves as a model system.

Purpose of the Study:

• To develop an atomistic model of the excited state ensemble of a stabilized CheY mutant.
• To characterize the structural features of transient protein excited states.
• To demonstrate a hybrid simulation-experimental approach for studying protein dynamics.

Main Methods:

• Utilized 42 milliseconds of all-atom molecular dynamics simulations as a prior.
• Refined the simulation data against small-angle X-ray scattering (SAXS) data using the EROS method.
• Integrated simulation and experimental data to build a statistical model of the excited state ensemble.

Main Results:

• Developed an atomistic model of the excited state ensemble for CheY.
• Identified an unstructured N-terminus stabilized by non-native contacts in the excited state.
• The excited state conformation was topologically simpler than the native state.
• Predicted single molecule Förster Resonance Energy Transfer (smFRET) experiments for validation.

Conclusions:

• The study successfully combined simulation and experiment to model protein excited states.
• The findings provide insights into the structure and dynamics of transient protein conformations.
• This approach enables rational design of experiments to validate structural models of protein excited states.