Backbone rigidity of disordered protein linkers from NMR experiments and MD simulations

Area of Science:

• Biophysics
• Computational Biology
• Protein Engineering

Background:

• Disordered protein linkers are crucial for multidomain protein function and engineering.
• Quantitative biophysical characterization methods for these linkers are limited.

Purpose of the Study:

• To develop and validate a quantitative method for characterizing disordered protein linker biophysics.
• To investigate the relationship between backbone dynamics and rigidity in short disordered linkers.
• To assess the impact of amino acid composition and ionic strength on linker properties.

Main Methods:

• Combined Nuclear Magnetic Resonance (NMR) spectroscopy with molecular dynamics (MD) simulations.
• Utilized a tailored Quality Evaluation Based Simulation Selection (QEBSS) framework.
• Analyzed four model peptides representing common linker sequences: (GGS)3, (GPS)3, K(AP)5K, and a longer charged peptide.

Main Results:

• Protein backbone 15N spin relaxation times directly correlate with backbone rigidities in short disordered linkers.
• Glycine-rich linkers exhibited looping, while proline-rich linkers showed extended conformations with slower dynamics.
• Sodium and calcium binding had minimal impact on the rigidity of charged linkers, suggesting electrostatics are not the primary determinant of stiffness.

Conclusions:

• The integrated NMR and MD simulation approach provides accurate, quantitative insights into disordered linker properties.
• This method enhances the accuracy of MD simulations for disordered protein regions.
• Findings offer valuable biophysical understanding for protein design and the development of machine learning models in structural biology.