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

Best-practice IgM- and IgA-enriched immunoglobulin use in patients with sepsis

Background: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite treatment being in line with current guidelines, mortality remains high in those with septic shock. Intravenous immunoglobulins represent a promising therapy to modulate both the pro- and anti-inflammatory processes and can contribute to the elimination of pathogens. In this context, there is evidence of the benefits of immunoglobulin M (IgM)- and immunoglobulin A (IgA)-enriched immunoglobulin therapy for sepsis. This manuscript aims to summarize current relevant data to provide expert opinions on best practice for the use of an IgM- and IgA-enriched immunoglobulin (Pentaglobin) in adult patients with sepsis. Main text: Sepsis patients with hyperinflammation and patients with immunosuppression may benefit most from treatment with IgM- and IgA-enriched immunoglobulin (Pentaglobin). Patients with hyperinflammation present with phenotypes that manifest throughout the body, whilst the clinical characteristics of immunosuppression are less clear. Potential biomarkers for hyperinflammation include elevated procalcitonin, interleukin-6, endotoxin activity and C-reactive protein, although thresholds for these are not well-defined. Convenient biomarkers for identifying patients in a stage of immune-paralysis are still matter of debate, though human leukocyte antigen–antigen D related expression on monocytes, lymphocyte count and viral reactivation have been proposed. The timing of treatment is potentially more critical for treatment efficacy in patients with hyperinflammation compared with patients who are in an immunosuppressed stage. Due to the lack of evidence, definitive dosage recommendations for either population cannot be made, though we suggest that patients with hyperinflammation should receive an initial bolus at a rate of up to 0.6 mL (30 mg)/kg/h for 6 h followed by a continuous maintenance rate of 0.2 mL (10 mg)/kg/hour for ≥ 72 h (total dose ≥ 0.9 g/kg). For immunosuppressed patients, dosage is more conservative (0.2 mL [10 mg]/kg/h) for ≥ 72 h, without an initial bolus (total dose ≥ 0.72 g/kg). Conclusions: Two distinct populations that may benefit most from Pentaglobin therapy are described in this review. However, further clinical evidence is required to strengthen support for the recommendations given here regarding timing, duration and dosage of treatment

Theory of High-Force DNA Stretching and Overstretching

Single molecule experiments on single- and double stranded DNA have sparked a renewed interest in the force-extension of polymers. The extensible Freely Jointed Chain (FJC) model is frequently invoked to explain the observed behavior of single-stranded DNA. We demonstrate that this model does not satisfactorily describe recent high-force stretching data. We instead propose a model (the Discrete Persistent Chain, or ``DPC'') that borrows features from both the FJC and the Wormlike Chain, and show that it resembles the data more closely. We find that most of the high-force behavior previously attributed to stretch elasticity is really a feature of the corrected entropic elasticity; the true stretch compliance of single-stranded DNA is several times smaller than that found by previous authors. Next we elaborate our model to allow coexistence of two conformational states of DNA, each with its own stretch and bend elastic constants. Our model is computationally simple, and gives an excellent fit through the entire overstretching transition of nicked, double-stranded DNA. The fit gives the first values for the elastic constants of the stretched state. In particular we find the effective bend stiffness for DNA in this state to be about 10 nm*kbt, a value quite different from either B-form or single-stranded DNAComment: 33 pages, 11 figures. High-quality figures available upon reques

Mechanical Stability of a High-Affinity Toxin Anchor from the Pathogen Clostridium perfringens

The opportunistic pathogen Clostridium perfringens assembles its toxins and carbohydrate-active enzymes by the high-affinity cohesin-dockerin (Coh-Doc) interaction. Coh-Doc interactions characterized previously have shown considerable resilience towards mechanical stress. Here, we aimed to determine the mechanics of this interaction from C. perfringens in the context of a pathogen. Using atomic force microscopy based single-molecule force spectroscopy (AFM-SMFS) we probed the mechanical properties of the interaction of a dockerin from the $mu$-toxin with the GH84C X82 cohesin domain of C. perfringens. Most probable complex rupture forces were found to be approximately 60 pN. An estimate of the binding potential width was performed using two different methods of loading rate determination. The dockerin was expressed with its adjacent FIVAR (Found in Various Architectures) domain, whose mechanostability we determined to be very similar to the complex. Additionally, fast refolding of this domain was observed. The Coh-Doc interaction from C. perfringens is the mechanically weakest observed to date. Our results establish the relevant force range of toxin assembly mechanics in pathogenic Clostridia

Local buffer mechanisms for population persistence

Assessing and predicting the persistence of populations is essential for the conservation and control of species. Here, we argue that local mechanisms require a better conceptual synthesis to facilitate a more holistic consideration along with regional mechanisms known from metapopulation theory. We summarise the evidence for local buffer mechanisms along with their capacities and emphasise the need to include multiple buffer mechanisms in studies of population persistence. We propose an accessible framework for local buffer mechanisms that distinguishes between damping (reducing fluctuations in population size) and repelling (reducing population declines) mechanisms. We highlight opportunities for empirical and modelling studies to investigate the interactions and capacities of buffer mechanisms to facilitate better ecological understanding in times of ecological upheaval.acceptedVersio

Ultrastable cellulosome-adhesion complex tightens under load

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass

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