Single Molecule Study of the Intrinsically Disordered FG-Repeat Nucleoporin 153

AbstractNucleoporins (Nups), which are intrinsically disordered, form a selectivity filter inside the nuclear pore complex, taking a central role in the vital nucleocytoplasmic transport mechanism. These Nups display a complex and nonrandom amino-acid architecture of phenylalanine glycine (FG)-repeat clusters and intra-FG linkers. How such heterogeneous sequence composition relates to function and could give rise to a transport mechanism is still unclear. Here we describe a combined chemical biology and single-molecule fluorescence approach to study the large human Nup153 FG-domain. In order to obtain insights into the properties of this domain beyond the average behavior, we probed the end-to-end distance (RE) of several ∼50-residues long FG-repeat clusters in the context of the whole protein domain. Despite the sequence heterogeneity of these FG-clusters, we detected a reoccurring and consistent compaction from a relaxed coil behavior under denaturing conditions (RE/RE,RC = 0.99 ± 0.15 with RE,RC corresponding to ideal relaxed coil behavior) to a collapsed state under native conditions (RE/RE,RC = 0.79 ± 0.09). We then analyzed the properties of this protein on the supramolecular level, and determined that this human FG-domain was in fact able to form a hydrogel with physiological permeability barrier properties

Cargo transport through the nuclear pore complex at a glance

Bidirectional transport of macromolecules across the nuclear envelope is a hallmark of eukaryotic cells, in which the genetic material is compartmentalized inside the nucleus. The nuclear pore complex (NPC) is the major gateway to the nucleus and it regulates nucleocytoplasmic transport, which is key to processes including transcriptional regulation and cell cycle control. Accordingly, components of the nuclear transport machinery are often found to be dysregulated or hijacked in diseases. In this Cell Science at a Glance article and accompanying poster, we provide an overview of our current understanding of cargo transport through the NPC, from the basic transport signals and machinery to more emerging aspects, all from a 'cargo perspective'. Among these, we discuss the transport of large cargoes (>15 nm), as well as the roles of different cargo properties to nuclear transport, from size and number of bound nuclear transport receptors (NTRs), to surface and mechanical properties

Formal Scenario-Based Testing of Autonomous Vehicles: From Simulation to the Real World

We present a new approach to automated scenario-based testing of the safety of autonomous vehicles, especially those using advanced artificial intelligence-based components, spanning both simulation-based evaluation as well as testing in the real world. Our approach is based on formal methods, combining formal specification of scenarios and safety properties, algorithmic test case generation using formal simulation, test case selection for track testing, executing test cases on the track, and analyzing the resulting data. Experiments with a real autonomous vehicle at an industrial testing facility support our hypotheses that (i) formal simulation can be effective at identifying test cases to run on the track, and (ii) the gap between simulated and real worlds can be systematically evaluated and bridged.Comment: 9 pages, 6 figures. Full version of an ITSC 2020 pape

Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.We are grateful for helpful comments and various discussions with Cedric Debes, Martin Beck as well as the whole Lemke group. We thank Guillaume Bouvignies for help with relaxation dispersion experiments, and Damien Maurin for sample preparation. S.M. acknowledges funding from the Boehringer Ingelheim Fonds (BIF) and an EMBO long-term fellowship (ALTF 468-2014) and EC (EMBOCOFUND2012, GA-2012-600394) via Marie Curie Action. I.V.A. acknowledges a BIF short-term fellowship. J.C. and S.L.S. are supported by the Wellcome Trust. J.C. is a Wellcome Trust Senior Research Fellow (WT/095195). E.A.L. is grateful to funds from the SFB1129 and the Emmy Noether program of the DFG, F.G. from the Klaus Tschira Foundation, and D.M. from the BIOMS program of Heidelberg University. We are also grateful to instrument access via the EMBL Pepcore facility.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.cell.2015.09.04

Manipulating charge transfer excited state relaxation and spin crossover in iron coordination complexes with ligand substitution

Developing light-harvesting and photocatalytic molecules made with iron could provide a cost effective, scalable, and environmentally benign path for solar energy conversion. To date these developments have been limited by the sub-picosecond metal-to-ligand charge transfer (MLCT) electronic excited state lifetime of iron based complexes due to spin crossover-the extremely fast intersystem crossing and internal conversion to high spin metal-centered excited states. We revitalize a 30 year old synthetic strategy for extending the MLCT excited state lifetimes of iron complexes by making mixed ligand iron complexes with four cyanide (CN-;) ligands and one 2,2′-bipyridine (bpy) ligand. This enables MLCT excited state and metal-centered excited state energies to be manipulated with partial independence and provides a path to suppressing spin crossover. We have combined X-ray Free-Electron Laser (XFEL) Kβ hard X-ray fluorescence spectroscopy with femtosecond time-resolved UV-visible absorption spectroscopy to characterize the electronic excited state dynamics initiated by MLCT excitation of [Fe(CN)4(bpy)]2-. The two experimental techniques are highly complementary; the time-resolved UV-visible measurement probes allowed electronic transitions between valence states making it sensitive to ligand-centered electronic states such as MLCT states, whereas the Kβ fluorescence spectroscopy provides a sensitive measure of changes in the Fe spin state characteristic of metal-centered excited states. We conclude that the MLCT excited state of [Fe(CN)4(bpy)]2- decays with roughly a 20 ps lifetime without undergoing spin crossover, exceeding the MLCT excited state lifetime of [Fe(2,2′-bipyridine)3]2+ by more than two orders of magnitude