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.

Abstract:The Helium Ion Microscope (HIM) has the capability to image small features with a resolution down to 0.35 nm due to its highly focused gas field ionization source and its small beam-sample interaction volume. In this work, the focused helium ion beam of a HIM is utilized to create nanopores with diameters down to 1.3 nm. It will be demonstrated that nanopores can be milled into silicon nitride, carbon nanomembranes (CNMs) and graphene with well-defined aspect ratio. To image and characterize the produced nanopores, helium ion microscopy and high resolution scanning transmission electron microscopy were used. The analysis of the nanopores' growth behavior, allows inferring on the profile of the helium ion beam.Nanopores in atomically thin membranes can be used for biomolecule analysis, 1 electrochemical storage, 2 as well as for the separation of gases and liquids. 3 All of these applications require a precise control of the size and shape of the nanopores. It was shown that the focused beam of a transmission electron microscope (TEM) is able to create nanopores in membranes of silicon nitride and graphene with diameters down to 2 nm. 4,5 Pores can be further shrunk in a TEM by areal electron impact. 6 However, the preparation of such nanopores in a TEM is time-consuming and is limited to small samples (~3 mm diameter) that fit into the microscope. Focused ion beam tools (FIB) offer more flexibility concerning the sample size and a higher milling speed. Among these FIB tools gallium liquid metal ion sources (LMIS) are widely used, allowing minimum sizes of 3 nm diameter for nanopores. 7 The development of a reliable gas field ionization source (GFIS) type allowed the construction of the helium ion microscope which surpasses the imaging and milling resolution of 9 First studies about milling with helium ions reported sample damage by amorphization and helium implantation during milling on bulk substrates. 10 The latter effect is absent on membranes, where nanopores with diameters of 2.6 nm were milled by HIM. 11 In all these reports, pores were created by single spot exposures. Here we present a different route to create small nanopores in membranes by milling circular patterns. Furthermore we are able to connect the growth of nanopores to the ion beam profile.

Strong coupling between two quanta of different excitations leads to the formation of a hybridized state which paves a way for exploiting new degrees of freedom to control phenomena with high efficiency and precision. A magnon polaron is the hybridized state of a phonon and a magnon, the elementary quanta of lattice vibrations and spin waves in a magnetically-ordered material. A magnon polaron can be formed at the intersection of the magnon and phonon dispersions, where their frequencies coincide.The observation of magnon polarons in the time domain has remained extremely challenging because the weak interaction of magnons and phonons and their short lifetimes jeopardize the strong coupling required for the formation of a hybridized state. Here, we overcome these limitations by spatial matching of magnons and phonons in a metallic ferromagnet with a nanoscale periodic surface pattern.The spatial overlap of the selected phonon and magnon modes formed in the periodic ferromagnetic structure results in a high coupling strength which, in combination with their long lifetimes allows us to find clear evidence of an optically excited magnon polaron. We show that the symmetries of the localized magnon and phonon states play a crucial role in the magnon polaron formation and its manifestation in the optically excited magnetic transients.

Single stranded DNA (ssDNA) binding protein plays an important role in the DNA replication process in a wide range of organisms. It binds to ssDNA to prevent premature re annealing and to protect it from degradation. Current understanding of SSB/ssDNA interaction points to a complex mechanism, including SSB motion along the DNA strand. We report on the first characterization of this interaction at the single molecule level using solid state nanopore sensors, namely without any labelling or surface immobilisation. Our results show that the presence of SSB on the ssDNA can control the speed of nanopore translocation, presumably due to strong interactions between SSB and the nanopore surface. This enables nanopore based detection of ssDNA fragments as short as 37 nt, which is normally very difficult with solid state nanopore sensors, due to constraints in noise and bandwidth.Notably, this fragment is considerably shorter than the 65 nt binding motif, typically required for SSB binding at high salt concentrations. The non specificity of SSB binding to ssDNA further suggests that this approach could be used for fragment sizing of short ssDNA.

No abstract