Structural characterization of labeled clathrin and coated vesicles

Area of Science:

• Biochemistry
• Cell Biology
• Structural Biology

Background:

• Clathrin self-assembly forms coated vesicles, essential for intracellular trafficking.
• Understanding the molecular mechanisms of clathrin assembly is crucial for cell biology.

Purpose of the Study:

• To investigate the conformational changes in clathrin during self-assembly.
• To determine the role of these conformational changes in the initiation of coated vesicle formation.

Main Methods:

• Covalent labeling of clathrin and coated vesicles using N-(1-anilinonaphthalene)maleimide (AN).
• Fluorescence energy transfer measurements to detect conformational changes.
• pH-dependent self-association assays and coated vesicle recombination studies.
• Trypsin digestion and SDS-PAGE to analyze protein structure and integrity.

Main Results:

• A significant increase in fluorescence energy transfer was observed in clathrin at pH 6.0-6.5, coinciding with rapid self-association.
• This conformational rearrangement is proposed to initiate clathrin self-assembly into coat structures.
• Labeled clathrin readily reassembled into coated vesicles, indicating labeling did not impede function.
• Trypsin digestion revealed structural similarities between clathrin baskets and coated vesicles.

Conclusions:

• Clathrin undergoes a pH-dependent conformational change that triggers self-assembly.
• This process is essential for the formation of coated vesicles.
• The labeling method is suitable for studying clathrin assembly dynamics without compromising function.