ac3035025_si_001.pdf (363.15 kB)
Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores
journal contribution
posted on 2013-02-19, 00:00 authored by Deanpen Japrung, Jakob Dogan, Kevin J. Freedman, Achim Nadzeyka, Sven Bauerdick, Tim Albrecht, Min Jun Kim, Per Jemth, Joshua B. EdelPartially or fully disordered proteins are instrumental
for signal-transduction
pathways; however, many mechanistic aspects of these proteins are
not well-understood. For example, the number and nature of intermediate
states along the binding pathway is still a topic of intense debate.
To shed light on the conformational heterogeneity of disordered protein
domains and their complexes, we performed single-molecule experiments
by translocating disordered proteins through a nanopore embedded within
a thin dielectric membrane. This platform allows for single-molecule
statistics to be generated without the need of fluorescent labels
or other modification groups. These studies were performed on two
different intrinsically disordered protein domains, a binding domain
from activator of thyroid hormone and retinoid receptors (ACTR) and
the nuclear coactivator binding domain of CREB-binding protein (NCBD),
along with their bimolecular complex. Our results demonstrate that
both ACTR and NCBD populate distinct conformations upon translocation
through the nanopore. The folded complex of the two disordered domains,
on the other hand, translocated as one conformation. Somewhat surprisingly,
we found that NCBD undergoes a charge reversal under high salt concentrations.
This was verified by both translocation statistics as well as by measuring
the ζ-potential. Electrostatic interactions have been previously
suggested to play a key role in the association of intrinsically disordered
proteins, and the observed behavior adds further complexity to their
binding reactions.