Exploring conformational changes coupled to ionization states using a hybrid Rosetta-MCCE protocol.

Area of Science:

• Biophysics
• Computational Biology
• Protein Chemistry

Background:

• Estimating pK(a) values is crucial for understanding protein function and designing mutants.
• Standard methods like Multi-Conformation Continuum Electrostatics (MCCE) are limited by considering only sidechain conformational changes.

Purpose of the Study:

• To develop and validate a hybrid protocol combining Rosetta full-atom refinement and MCCE for improved pK(a) prediction.
• To assess the protocol's performance in blind prediction of pK(a) for mutated residues in Staphylococcal nuclease (SNase).

Main Methods:

• A hybrid protocol integrating Rosetta refinement (for backbone changes) and MCCE (for electrostatics) was employed.
• The protocol combined Rosetta's non-electrostatic energy with MCCE's electrostatic energy to calculate ionization states.
• Applied to blind prediction of 94 mutated residues in SNase as part of a benchmark test.

Main Results:

• The hybrid Rosetta/MCCE protocol achieved a root mean squared deviation (RMSD) of 4.3 for 63 measured pK(a)s, outperforming standard MCCE (RMSD 6.6).
• The protocol reduced the pK(a) shift (ΔpK(a)) by optimizing electrostatic interactions and lowering desolvation penalties.
• Analysis indicated that the protocol samples conformations yielding pK(a) values closer to experimental measurements.

Conclusions:

• The hybrid Rosetta/MCCE protocol offers a significant improvement in pK(a) prediction accuracy by incorporating backbone conformational changes.
• The study highlights the importance of considering both electrostatic and non-electrostatic contributions, including desolvation effects.
• Accurate estimation of reorganization energy remains a challenge for future protocol development.