Multistep protein unfolding during nanopore translocation

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Cellular compartments are enclosed by lipid bilayer membranes.
• Transport of molecules across membranes is essential for cell function.
• Understanding protein translocation across membranes is crucial for cellular processes.

Purpose of the Study:

• To investigate the mechanism of folded protein translocation through transmembrane pores.
• To examine co-translocational unfolding of individual protein molecules.
• To elucidate the pathway of protein movement between cellular compartments.

Main Methods:

• Utilized a model protein nanopore system.
• Tagged protein substrates with oligonucleotides for potential-driven movement.
• Analyzed the process of co-translocational unfolding using a DNA tag.
• Compared the observed unfolding pathway with force spectroscopy and solution denaturation methods.

Main Results:

• A four-step translocation mechanism was supported for model thioredoxin substrates.
• The process involves DNA tag capture, pore-induced local unfolding, spontaneous unfolding, and diffusion.
• Co-translocational unfolding was observed for individual protein molecules.
• The elucidated unfolding pathway differs from two-state mechanisms observed in solution denaturation.

Conclusions:

• A novel four-step mechanism describes protein translocation and unfolding through nanopores.
• This mechanism provides new insights into how proteins navigate membrane barriers.
• The findings challenge existing models of protein unfolding and translocation.