Single-molecule studies of intrinsically disordered proteins using solid-state nanopores.

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Partially or fully disordered proteins are crucial for signal-transduction but their mechanisms remain unclear.
• The number and nature of intermediate states in protein binding pathways are debated.
• Understanding conformational heterogeneity is key to elucidating disordered protein function.

Purpose of the Study:

• To investigate the conformational heterogeneity of intrinsically disordered protein domains (ACTR and NCBD) and their complex.
• To explore the binding pathways and conformational dynamics of these proteins using single-molecule experiments.
• To understand the role of electrostatic interactions in the binding of disordered proteins.

Main Methods:

• Single-molecule experiments involving translocation of disordered proteins through a nanopore.
• Utilizing a nanopore platform for label-free, single-molecule statistics generation.
• Measuring translocation statistics and ζ-potential to analyze protein behavior and charge.

Main Results:

• Both ACTR and NCBD proteins populated distinct conformations during nanopore translocation.
• The complex of ACTR and NCBD translocated as a single conformational state.
• NCBD exhibited a surprising charge reversal under high salt concentrations, verified by translocation data and ζ-potential measurements.

Conclusions:

• Disordered proteins possess significant conformational heterogeneity that influences their function.
• The observed charge reversal of NCBD highlights the complex role of electrostatics in disordered protein interactions.
• Nanopore translocation offers a powerful label-free method for studying disordered protein dynamics.