Dissecting In Vivo Cross-Linker Performance: Insights into Structural Dynamics and Molecular Rigidity

Area of Science:

• Biochemistry
• Structural Biology
• Proteomics

Background:

• Understanding protein structure and dynamics is vital for biological function.
• In vitro methods often miss in vivo complexities.
• AI tools like AlphaFold improve structure determination but not in vivo dynamics.

Purpose of the Study:

• To evaluate in vivo chemical cross-linking mass spectrometry (XL-MS) for studying protein structure and dynamics.
• To analyze the performance of four common cross-linkers based on their structural and dynamic properties.
• To establish a framework for selecting and designing cross-linkers for in vivo XL-MS.

Main Methods:

• Systematic evaluation of four cross-linkers using in vivo XL-MS.
• Analysis of cross-linker structural characteristics (length, dihedral angles).
• Integration of in vivo XL-MS data with all-atom molecular dynamics simulations.

Main Results:

• Established the relationship between cross-linker flexibility, structural characteristics, and effectiveness in probing protein dynamics.
• Demonstrated that more rigid cross-linkers capture more dynamic information.
• Highlighted the enhanced ability of rigid cross-linkers to capture dynamics in disordered protein regions.

Conclusions:

• Cross-linker design is critical for effective in vivo XL-MS.
• Rigid cross-linkers offer advantages for studying protein dynamics in native cellular environments.
• This work provides a foundation for advancing in vivo XL-MS applications in cell biology.