title: 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.
authors: Department of Chemistry, Imperial College London, South Kensington, SW7 2AZ, London, United Kingdom.
authors: Deanpen
authors: D
authors: Japrung
authors: Jakob
authors: J
authors: Dogan
authors: Kevin J
authors: KJ
authors: Freedman
authors: Achim
authors: A
authors: Nadzeyka
authors: Sven
authors: S
authors: Bauerdick
authors: Tim
authors: T
authors: Albrecht
authors: Min Jun
authors: MJ
authors: Kim
authors: Per
authors: P
authors: Jemth
authors: Joshua B
authors: JB
authors: Edel
mesh_terms: D050882:CREB-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008027:Light; D058608:Nanopores; D056921:Nuclear Receptor Coactivator 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012492:Salts / Q000737:chemistry; D012542:Scattering, Radiation; D015398:Signal Transduction; D055672:Static Electricity; D013963:Thyroid Hormones / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012492:Salts; D013963:Thyroid Hormones; D050882:CREB-Binding Protein; C538866:NCOA3 protein, human; D056921:Nuclear Receptor Coactivator 3