Mapping of Protein Interfaces in Live Cells Using Genetically Encoded Crosslinkers

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Protein-protein interactions are crucial in biomedicine, but traditional methods like X-ray crystallography are limited to artificial environments.
• Investigating protein interactions in a physiological context and characterizing elusive complexes requires alternative approaches.

Purpose of the Study:

• To develop a general strategy for investigating protein interactions at the molecular level directly within live mammalian cells.
• To map interaction surfaces and determine spatial constraints for accurate molecular modeling of protein complexes.

Main Methods:

• Genetic incorporation of photo- and chemical crosslinking noncanonical amino acids.
• Utilizing photo-crosslinking amino acids to map protein proximity and identify interaction surfaces.
• Employing chemical crosslinkers to define inter-molecular spatial constraints for modeling.

Main Results:

• Demonstrated a strategy for mapping protein-protein interactions in live cells using noncanonical amino acids.
• Successfully applied the method to determine the binding of Urocortin1 to its receptor, corticotropin releasing factor receptor type 1.
• Established a technique for obtaining inter-molecular spatial constraints to build accurate molecular models.

Conclusions:

• The described strategy enables molecular-level investigation of protein interactions directly in live mammalian cells.
• This approach is versatile, applicable to various protein complexes, and utilizes established techniques.
• The method provides a powerful tool for structural biology and molecular modeling in a physiological context.