Pan1 is an intrinsically disordered protein with homotypic interactions

Area of Science:

• Cell biology
• Protein biochemistry

Background:

• The yeast scaffold protein Pan1 is crucial for endocytosis.
• Pan1 possesses N-terminal EH domains, a central coiled-coil region, and a C-terminal proline-rich domain.
• Intrinsically disordered regions suggest diverse protein interactions.

Purpose of the Study:

• To investigate the homotypic interaction and dimerization of Pan1.
• To characterize the conformational states and dynamics of Pan1 using tryptophan fluorescence.
• To elucidate the role of specific tryptophan residues in Pan1 structure and function.

Main Methods:

• In vitro biochemical assays to identify dimerization interfaces.
• Site-directed mutagenesis to create single-tryptophan Pan1 mutants.
• Tryptophan fluorescence quenching (acrylamide, iodide, cesium) to probe local environments.
• Time-resolved fluorescence anisotropy to assess protein dynamics.

Main Results:

• Amino acids 705-848 were identified as critical for Pan1 homotypic interaction and dimerization.
• Single-tryptophan mutants exhibited collisional quenching, indicating moderate accessibility.
• Differential Stern-Volmer constants with iodide and cesium revealed unique electrostatic environments for each tryptophan.
• Fluorescence anisotropy data supported structural and disorder predictions.

Conclusions:

• Pan1 exists as a dimer, with a defined interface.
• Tryptophan fluorescence is a valuable tool for characterizing Pan1's local environments and conformational dynamics.
• Understanding Pan1 dynamics is key to deciphering endocytosis mechanisms.