Single-Molecule Studies of Intrinsically Disordered Proteins Using Solid-State Nanopores

Abstract

Partially 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.

SSB Binding to Single-Stranded DNA Probed Using Solid-State Nanopore Sensors

“…3a. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 adsorption to the inner pore walls indeed plays a significant role during the translocation process, as reported previously for individual protein species, 17 then the translocation time would be greatly affected by adsorption and desorption of ssDNA bound SSB inside the pore.…”

A Folded Excited State of Ligand-Free Nuclear Coactivator Binding Domain (NCBD) Underlies Plasticity in Ligand Recognition

“…These experiments revealed three different translocation events corresponding to three different conformations. 16 Because of its small size and dynamics, NCBD is an optimal model system for computational studies of flexible molecular recognition. The time scales of folding and large scale conformational reconfigurations of NCBD are still beyond the reach of all-atom simulations.…”

Electrodeposition and Bipolar Effects in Metallized Nanopores and Their Use in the Detection of Insulin

“…56 However, enhancing protein adsorption inside the nanopore has been shown to be viable strategy for increasing the translocation time. 3,21,57 Therefore, we modified the inside of the pore with homocysteine (Hcy). Hcy binds to the Au to form a strong Au-thiol bond and exhibits two additional functional groups, namely, an amino-and carboxyl group at the α-carbon.…”

“…S olid-state nanopores have been successfully employed as versatile sensing devices for detection of single analytes such as DNA, 1,2 proteins, 3 and gold nanoparticles. 4 The major technological goal in the field has been the construction of fast and cheap nanopore-based next-generation DNA sequencers.…”