Single-molecule protein unfolding in solid state nanopores

Area of Science:

• Biophysics
• Nanotechnology
• Protein Science

Background:

• Understanding protein folding and unfolding is crucial for comprehending biological functions.
• Single-molecule techniques offer high resolution for studying complex biological processes like protein translocation.
• Nanopore technology provides a platform for analyzing individual molecules.

Purpose of the Study:

• To investigate the folding states of single proteins using silicon nitride nanopores.
• To measure the excluded volumes of proteins during translocation to infer their conformational states.
• To explore the influence of electrical forces on protein unfolding within nanopores.

Main Methods:

• Utilized single silicon nitride nanopore devices for translocation measurements.
• Calibrated translocation signals using DNA to determine excluded volumes.
• Analyzed translocation signals of beta-lactoglobulin and histidine-containing phosphocarrier protein.
• Applied physiologically relevant electrical potentials to induce protein unfolding.

Main Results:

• Excluded volume measurements quantitatively matched predictions based on amino acid partial volumes for stalled translocations.
• The majority of translocating proteins were found to be in linear or looped conformations.
• Electrical forces were demonstrated to be capable of unfolding proteins under relevant conditions.
• Nanopore translocation signals proved sensitive enough to differentiate protein folding states.

Conclusions:

• Single nanopore translocation measurements can accurately determine protein excluded volumes and infer folding states.
• Protein translocation dynamics are influenced by intrinsic charge sequences and external electrical fields.
• Nanopore analysis offers a powerful tool for studying protein conformational changes and electrical unfolding mechanisms.