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In recent years, J. R. Kider and Z.A. Hussain have introduced the notion of standard fuzzy metric space and some basic properties were proved. In this paper, we generalize this notion to the setting of single valued neutrosophic sets. Furthermore, some interesting properties related to this notion are studied such as the continuity property of the mappings defined on standard single valued neutrosophic metric spaces. As an application, we establish the Edelstein fixed point theorem for standard single valued neutrosophic metric spaces. [ABSTRACT FROM AUTHOR]

In this paper, we introduce and study the notions of prime, maximal and principal single-valued neutrosophic ideals (resp. filters) on a lattice. Several properties and characterizations of these types of ideals and filters are given, and relationships between them are discussed. [ABSTRACT FROM AUTHOR]

In this paper, we generalize the notion of principal ideal (resp. filter) on a lattice to the setting of intuitionistic fuzzy sets and investigate their various characterizations and properties. More specifically, we show that any principal intuitionistic fuzzy ideal (resp. filter) coincides with an intuitionistic fuzzy down-set (resp. up-set) generated by an intuitionistic fuzzy singleton. Afterwards, for a given intuitionistic fuzzy set, we introduce two intuitionistic fuzzy sets: its intuitionistic fuzzy down-set and up-set, and we investigate their interesting properties.

In this work, the fabrication of multifunctional periodic microstructures on pure aluminium is presented. Three different geometries were fabricated with feature sizes ranging between 7 µm and 50 µm by using laser-based microstructuring methods. In detail, nanosecond pulsed direct laser writing and picosecond pulsed direct laser interference patterning were used with infrared laser radiation. The wetting characteristics of these structures were investigated performing static water contact angle measurements as well as by measuring the contact angle hysteresis and the sliding angle. The final wetting results show constant static contact angles above 150°, permitting the water droplets to roll off the substrate as well as collecting contamination at the same time. This self-cleaning effect led to a reduction of up to 94% of the spread of 1 µm sized manganese oxide particles. In addition, the freezing time required for droplets laying on the patterned surfaces was increased nearly by 300% at a temperature of 20 °C below zero. Finally, the results are compared to finite element simulations of heat transfer.

The early phases of clathrin mediated endocytosis are organized through a highly complex interaction network mediated by clathrin associated sorting proteins (CLASPs) that comprise long intrinsically disordered regions (IDRs). AP180 is a CLASP exclusively expressed in neurons and comprises a long IDR of around 600 residues, whose function remains partially elusive. Using NMR spectroscopy, we discovered an extended and strong interaction site within AP180 with the major adaptor protein AP2, and describe its binding dynamics at atomic resolution. We find that the 70 residue-long site determines the overall interaction between AP180 and AP2 in a dynamic equilibrium between its bound and unbound states, while weaker binding sites contribute to the overall affinity at much higher concentrations of AP2. Our data suggest that this particular interaction site might play a central role in recruitment of adaptors to the clathrin coated pit, whereas more transient and promiscuous interactions allow reshaping of the interaction network until cargo uptake inside a coated vesicle., (© 2024. The Author(s).)

Many proteins require different structural states or conformations for function, and intrinsically disordered proteins, i.e. proteins without stable three-dimensional structure, are certainly an extreme. Single molecule fluorescence and nuclear magnetic resonance (NMR) spectroscopy are both exceptionally well suited to decipher and describe these states and their interconversion. Different time scales, from picoseconds to several milliseconds, can be addressed by both techniques. The length scales probed and the sample requirements (e.g. concentration, molecular weight, sample complexity) are, however, vastly different, making NMR and single molecule fluorescence an excellent combination for integrated studies. Here, we review recently undertaken approaches for the combined use of NMR and single molecule fluorescence to study protein dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.

Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions. We shed light on how these two techniques were employed synergistically for multidomain proteins harboring intrinsically disordered linkers, for veritable IDPs, but also for liquid-liquid phase separated systems. Additionally, we provide insights into the first approaches to use single molecule Förster resonance energy transfer (FRET) and NMR for the description of multiconformational models of IDPs.

Studying the conformational landscape of intrinsically disordered and partially folded proteins is challenging and only accessible to a few solution state techniques, such as nuclear magnetic resonance (NMR), small-angle scattering techniques, and single-molecule Förster resonance energy transfer (smFRET). While each of the techniques is sensitive to different properties of the disordered chain, such as local structural propensities, overall dimension, or intermediate- and long-range contacts, conformational ensembles describing intrinsically disordered proteins (IDPs) accurately should ideally respect all of these properties. Here we develop an integrated approach using a large set of FRET efficiencies and fluorescence lifetimes, NMR chemical shifts, and paramagnetic relaxation enhancements (PREs), as well as small-angle X-ray scattering (SAXS) to derive quantitative conformational ensembles in agreement with all parameters. Our approach is tested using simulated data (five sets of PREs and 15 FRET efficiencies) and validated experimentally on the example of the disordered domain of measles virus phosphoprotein, providing new insights into the conformational landscape of this viral protein that comprises transient structural elements and is more compact than an unfolded chain throughout its length. Rigorous cross-validation using FRET efficiencies, fluorescence lifetimes, and SAXS demonstrates the predictive nature of the calculated conformational ensembles and underlines the potential of this strategy in integrative dynamic structural biology.

Refractive index modification by laser micro-structuration of diffractive optical devices in ophthalmic polymers has recently been applied for refractive correction in the fields of optics and ophthalmology. In this work, Safrofilcon-A hydrogel, used as soft contact lenses, was processed by direct laser interference patterning (DLIP) to fabricate linear periodic patterns on the surface of the samples. Periodic modulation of the surface was attained under two-beam interference by using a Q-switched laser source with emission at 263 nm and 4 ns pulse duration. Features of processed areas were studied as a function of both the interference spatial period and the laser fluence. Optical confocal microscopy used to evaluate the topography of the processed samples showed that both structured height and surface roughness increased with laser fluence. Static water contact angle (WCA) measurements were carried out with deionized water droplets on the structured areas to evaluate the hydration properties of DLIP structures. It was observed that the laser structured areas induced a delay in the hydration process. Finally, microstructural changes induced in the structured areas were assessed by confocal micro-Raman spectroscopy showing that at low laser fluences the polymer structure remained almost unaltered. In addition, Raman spectra of hydrated samples recovered the original shape of areas structured at low laser fluence.

Ice-building up on the leading edge of wings and other surfaces exposed to icing atmospheric conditions can negatively influence the aerodynamic performances of aircrafts. In the past, research activities focused on understanding icing phenomena and finding effective countermeasures. Efforts have been dedicated to creating coatings capable of reducing the adhesion strength of ice to a surface. Nevertheless, coatings still lack functional stability, and their application can be harmful to health and the environment. Pulsed laser surface treatments have been proven as a viable technology to induce icephobicity on metallic surfaces. However, a study aimed to find the most effective microstructures for reducing ice adhesion still needs to be carried out. This study investigates the variation of the ice adhesion strength of micro-textured aluminum surfaces treated using laser-based methods. The icephobic performance is tested in an icing wind tunnel, simulating realistic icing conditions. Finally, it is shown that optimum surface textures lead to a reduction of the ice adhesion strength from originally 57 kPa down to 6 kPa, corresponding to a relative reduction of ~90%. Consequently, these new insights will be of great importance in the development of functionalized surfaces, permitting an innovative approach to prevent the icing of aluminum components.

Laser-microtextured surfaces have gained an increasing interest due to their enormous spectrum of applications and industrial scalability. Direct laser interference patterning (DLIP) and the well-established direct laser writing (DLW) methods are suitable as a powerful combination for the fabrication of single (DLW or DLIP) and multi-scale (DLW+DLIP) textures. In this work, four-beam DLIP and DLW were used independently and combined to produce functional textures on aluminum. The influence of the laser processing parameters, such as the applied laser fluence and the number of pulses, on the resulting topography was analyzed by confocal microscopy and scanning electron microscopy. The static long-term and dynamic wettability characteristics of the laser-textured surfaces were determined through water contact angle and hysteresis measurements, revealing superhydrophobic properties with static contact angles up to 163° and hysteresis as low as 9°. The classical Cassie-Baxter and Wenzel models were applied, permitting a deeper understanding of the observed wetting behaviors. Finally, mechanical stability tests revealed that the DLW elements in the multi-scale structure protects the smaller DLIP features under tribological conditions.

In this study, two-step approaches to fabricate periodic microstructures on polyethylene terephthalate (PET) and poly(methyl methacrylate) (PMMA) substrates are presented to control the wettability of polymeric surfaces. Micropillar arrays with periods between 1.6 and 4.6 µm are patterned by plate-to-plate hot embossing using chromium stamps structured by four-beam Direct Laser Interference Patterning (DLIP). By varying the laser parameters, the shape, spatial period, and structure height of the laser-induced topography on Cr stamps are controlled. After that, the wettability properties, namely the static, advancing/receding contact angles (CAs), and contact angle hysteresis were characterized on the patterned PET and PMMA surfaces. The results indicate that the micropillar arrays induced a hydrophobic state in both polymers with CAs up to 140° in the case of PET, without modifying the surface chemistry. However, the structured surfaces show high adhesion to water, as the droplets stick to the surfaces and do not roll down even upon turning the substrates upside down. To investigate the wetting state on the structured polymers, theoretical CAs predicted by Wenzel and Cassie-Baxter models for selected structured samples with different topographical characteristics are also calculated and compared with the experimental data.

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.

The measles virus replication complex represents a potentially important, but as yet relatively unexplored target for viral inhibition. Little is known about the molecular mechanisms that underpin replication and transcription in paramyxoviruses. In recent years it has become clear that conformational dynamics play an important role in paramyxoviral replication, and that a complete understanding of the viral cycle requires a description of the structural plasticity of the different components. Here, we review recent progress in this direction, covering the dynamics of the nucleocapsid assembly process, high resolution structure and dynamics of protein:RNA interactions, and the investigation of the role of intrinsic conformational disorder in pre-assembly nucleoprotein/phosphoprotein complexes. Finally, we discuss the role of viral factories in the form of phase-separated membraneless organelles formed by measles virus phospho and nucleoproteins that promote the assembly of nucleocapsid structures., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Many viruses are known to form cellular compartments, also called viral factories. Paramyxoviruses, including measles virus, colocalize their proteomic and genomic material in puncta in infected cells. We demonstrate that purified nucleoproteins (N) and phosphoproteins (P) of measles virus form liquid-like membraneless organelles upon mixing in vitro. We identify weak interactions involving intrinsically disordered domains of N and P that are implicated in this process, one of which is essential for phase separation. Fluorescence allows us to follow the modulation of the dynamics of N and P upon droplet formation, while NMR is used to investigate the thermodynamics of this process. RNA colocalizes to droplets, where it triggers assembly of N protomers into nucleocapsid-like particles that encapsidate the RNA. The rate of encapsidation within droplets is enhanced compared to the dilute phase, revealing one of the roles of liquid-liquid phase separation in measles virus replication., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Intrinsically disordered proteins (IDPs) are flexible biomolecules whose essential functions are defined by their dynamic nature. Nuclear magnetic resonance (NMR) spectroscopy is ideally suited to the investigation of this behavior at atomic resolution. NMR relaxation is increasingly used to detect conformational dynamics in free and bound forms of IDPs under conditions approaching physiological, although a general framework providing a quantitative interpretation of these exquisitely sensitive probes as a function of experimental conditions is still lacking. Here, measuring an extensive set of relaxation rates sampling multiple-time-scale dynamics over a broad range of crowding conditions, we develop and test an integrated analytical description that accurately portrays the motion of IDPs as a function of the intrinsic properties of the crowded molecular environment. In particular we observe a strong dependence of both short-range and long-range motional time scales of the protein on the friction of the solvent. This tight coupling between the dynamic behavior of the IDP and its environment allows us to develop analytical expressions for protein motions and NMR relaxation properties that can be accurately applied over a vast range of experimental conditions. This unified dynamic description provides new insight into the physical behavior of IDPs, extending our ability to quantitatively investigate their conformational dynamics under complex environmental conditions, and accurately predicting relaxation rates reporting on motions on time scales up to tens of nanoseconds, both in vitro and in cellulo .

Fabricating aluminium surfaces with superhydrophobic and ice-repellent properties present nowadays a challenging task. In this work, multifunctional structures are manufactured by direct laser writing and direct laser interference patterning methods using pulsed infrared laser radiation (1064 nm). Different periodic patterns with feature sizes ranging from 7.0 to 50.0 µm are produced. In addition, hierarchical textures are produced combining both mentioned laser based methods. Water contact angle tests at room temperature showed that all produced patterns reached the superhydrophobic state after 13 to 16 days. In addition, these experiments were repeated at substrate temperatures from -30 °C to 80 °C allowing to determine three wettability behaviours as a function of the temperature. The patterned surfaces also showed ice-repellent properties characterized by a near three-fold increase in the droplets freezing times compared to the untreated samples. Using finite element simulations, it was found that the main reason behind the ice-prevention is the change in the droplet geometrical shape due to the hydrophobic nature of the treated surfaces. Finally, dynamic tests of droplets imping the treated aluminium surfaces cooled down to -20 °C revealed that only on the hierarchically patterned surface, the droplets were able to bounce off the substrate.

Measles virus is a negative strand virus and the genomic and antigenomic RNA binds to the nucleoprotein (N), assembling into a helical nucleocapsid. The polymerase complex comprises two proteins, the Large protein (L), that both polymerizes RNA and caps the mRNA, and the phosphoprotein (P) that co-localizes with L on the nucleocapsid. This review presents recent results about N and P, in particular concerning their intrinsically disordered domains. N is a protein of 525 residues with a 120 amino acid disordered C-terminal domain, N tail . The first 50 residues of N tail extricate the disordered chain from the nucleocapsid, thereby loosening the otherwise rigid structure, and the C-terminus contains a linear motif that binds P. Recent results show how the 5' end of the viral RNA binds to N within the nucleocapsid and also show that the bases at the 3' end of the RNA are rather accessible to the viral polymerase. P is a tetramer and most of the protein is disordered; comprising 507 residues of which around 380 are disordered. The first 37 residues of P bind N, chaperoning against non-specific interaction with cellular RNA, while a second interaction site, around residue 200 also binds N. In addition, there is another interaction between C-terminal domain of P (XD) and N tail . These results allow us to propose a new model of how the polymerase binds to the nucleocapsid and suggests a mechanism for initiation of transcription.

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly., Competing Interests: The authors declare no conflict of interest.

Over the last two decades, it has become increasingly clear that a large fraction of the human proteome is intrinsically disordered or contains disordered segments of significant length. These intrinsically disordered proteins (IDPs) play important regulatory roles throughout biology, underlining the importance of understanding their conformational behavior and interaction mechanisms at the molecular level. Here we review recent progress in the NMR characterization of the structure and dynamics of IDPs in various functional states and environments. We describe the complementarity of different NMR parameters for quantifying the conformational propensities of IDPs in their isolated and phosphorylated states, and we discuss the challenges associated with obtaining structural models of dynamic protein-protein complexes involving IDPs. In addition, we review recent progress in understanding the conformational behavior of IDPs in cell-like environments such as in the presence of crowding agents, in membrane-less organelles and in the complex environment of the human cell., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Measles virus genome encapsidation is essential for viral replication and is controlled by the intrinsically disordered phosphoprotein (P) maintaining the nucleoprotein in a monomeric form (N) before nucleocapsid assembly. All paramyxoviruses harbor highly disordered amino-terminal domains (P NTD ) that are hundreds of amino acids in length and whose function remains unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we describe the structure and dynamics of the 90-kDa N 0 P NTD complex, comprising 450 disordered amino acids, at atomic resolution. NMR relaxation dispersion reveals the existence of an ultraweak N-interaction motif, hidden within the highly disordered P NTD , that allows P NTD to rapidly associate and dissociate from a specific site on N while tightly bound at the amino terminus, thereby hindering access to the surface of N. Mutation of this linear motif quenches the long-range dynamic coupling between the two interaction sites and completely abolishes viral transcription/replication in cell-based minigenome assays comprising integral viral replication machinery. This description transforms our understanding of intrinsic conformational disorder in paramyxoviral replication. The essential mechanism appears to be conserved across Paramyxoviridae, opening unique new perspectives for drug development against this family of pathogens.

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Influenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility. This tail contains the Nuclear Location Signal (NLS). The nuclear trafficking of the viral components mobilizes cellular import factors at different stages, making these host-pathogen interactions promising targets for new therapeutics. NP is imported into the nucleus by the importin-α/β pathway, through a direct interaction with importin-α isoforms. Here we provide a combined nuclear magnetic resonance and small-angle X-ray scattering (NMR/SAXS) analysis to describe the dynamics of the interaction between influenza B NP and the human importin-α. The NP of influenza B does not have a single NLS nor a bipartite NLS but our results suggest that the tail harbors several adjacent NLS sequences, located between residues 30 and 71.

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration ( R G ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance ( R E ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values R G and R E For chemically denatured proteins we obtain mutual consistency in our inferences based on R G and R E , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between R E and R G that is amplified in the absence of denaturants. Therefore, joint assessments of R G and R E combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles., Competing Interests: The authors declare no conflict of interest.

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

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., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Understanding the function of intrinsically disordered proteins is intimately related to our capacity to correctly sample their conformational dynamics. So far, a gap between experimentally and computationally derived ensembles exists, as simulations show overcompacted conformers. Increasing evidence suggests that the solvent plays a crucial role in shaping the ensembles of intrinsically disordered proteins and has led to several attempts to modify water parameters and thereby favor protein-water over protein-protein interactions. This study tackles the problem from a different perspective, which is the use of the Kirkwood-Buff theory of solutions to reproduce the correct conformational ensemble of intrinsically disordered proteins (IDPs). A protein force field recently developed on such a basis was found to be highly effective in reproducing ensembles for a fragment from the FG-rich nucleoporin 153, with dimensions matching experimental values obtained from small-angle X-ray scattering and single molecule FRET experiments. Kirkwood-Buff theory presents a complementary and fundamentally different approach to the recently developed four-site TIP4P-D water model, both of which can rescue the overcollapse observed in IDPs with canonical protein force fields. As such, our study provides a new route for tackling the deficiencies of current protein force fields in describing protein solvation.

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Intrinsically disordered and phenylalanine-glycine-rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high-resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints., (Copyright © 2013 WILEY Periodicals, Inc.)

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)