Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein

The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in cooperation with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accelerate the folding of the multi-domain protein firefly luciferase (FLuc) 20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically-trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions. The Hsp70 system prevents protein aggregation and increases folding yields, but it is unknown whether it also enhances the rate of folding. Here the authors combine refolding assays, FRET and hydrogen/deuterium exchange-mass spectrometry measurements to study the folding of firefly luciferase and find that the bacterial Hsp70 actively promotes the folding of this multi-domain protein

Review

Molecular chaperones are highly conserved proteins that promote proper folding of other proteinsin vivo. Diverse chaperone systems assistde novoprotein folding and trafficking, the assembly of oligomeric complexes, and recovery from stress-induced unfolding. A fundamental function of molecular chaperones is to inhibit unproductive protein interactions by recognizing and protecting hydrophobic surfaces that are exposed during folding or following proteotoxic stress. Beyond this basic principle, it is now clear that chaperones can also actively and specifically accelerate folding reactions in an ATP-dependent manner. We focus on the bacterial Hsp70 and chaperonin systems as paradigms, and review recent work that has advanced our understanding of how these chaperones act as catalysts of protein folding

Novel Method to Process Cystic Fibrosis Sputum for Determination of Oxidative State

Background: Induced sputum is the most commonly used method to analyze airway inflammation in cystic fibrosis (CF) patients ex vivo. Due to the complex matrix of the sample material, precise and reliable analysis of sputum constituents depends critically on preanalytical issues. Objectives: Here we compared the commonly used method for sputum processing by dithiothreitol (DTT) with a novel mechanical method in regard to basal cellular parameters, neutrophil markers and glutathione (GSH) levels. Methods: Sputum samples from CF patients were processed in parallel with or without the use of DTT. The key improvement of the mechanical method was the processing in many very small aliquots. Cellular and humoral markers were assessed and compared according to Bland-Altman. Results: Total cell count, cell viability, differential cell count, neutrophil elastase levels and flow cytometrically analyzed neutrophil markers (CD63, CD11b, DHR) did not differ between the two methods. Intracellular and extracellular GSH levels were significantly higher in DTT-treated samples (p = 0.002). Conclusion: The mechanical sputum-processing method presented had a similar yield of cells and fluids as the conventional DTT method and the advantage of omitting the introduction of reducing agents. This method allows a more reliable analysis of redox-dependent airway inflammation in sputum cells and fluid from CF patients than methods utilizing DTT. Copyright (C) 2009 S. Karger AG, Base

Error threshold in the evolution of diploid organisms

The effects of error propagation in the reproduction of diploid organisms are studied within the populational genetics framework of the quasispecies model. The dependence of the error threshold on the dominance parameter is fully investigated. In particular, it is shown that dominance can protect the wild-type alleles from the error catastrophe. The analysis is restricted to a diploid analogue of the single-peaked landscape.Comment: 9 pages, 4 Postscript figures. Submitted to J. Phy. A: Mat. and Ge

Spinning test particles and clock effect in Kerr spacetime

We study the motion of spinning test particles in Kerr spacetime using the Mathisson-Papapetrou equations; we impose different supplementary conditions among the well known Corinaldesi-Papapetrou, Pirani and Tulczyjew's and analyze their physical implications in order to decide which is the most natural to use. We find that if the particle's center of mass world line, namely the one chosen for the multipole reduction, is a spatially circular orbit (sustained by the tidal forces due to the spin) then the generalized momentum $P$ of the test particle is also tangent to a spatially circular orbit intersecting the center of mass line at a point. There exists one such orbit for each point of the center of mass line where they intersect; although fictitious, these orbits are essential to define the properties of the spinning particle along its physical motion. In the small spin limit, the particle's orbit is almost a geodesic and the difference of its angular velocity with respect to the geodesic value can be of arbitrary sign, corresponding to the spin-up and spin-down possible alignment along the z-axis. We also find that the choice of the supplementary conditions leads to clock effects of substantially different magnitude. In fact, for co-rotating and counter-rotating particles having the same spin magnitude and orientation, the gravitomagnetic clock effect induced by the background metric can be magnified or inhibited and even suppressed by the contribution of the individual particle's spin. Quite surprisingly this contribution can be itself made vanishing leading to a clock effect undistiguishable from that of non spinning particles. The results of our analysis can be observationally tested.Comment: IOP macros, eps figures n. 12, to appear on Classical and Quantum Gravity, 200

Orbital evolution of a particle around a black hole: II. Comparison of contributions of spin-orbit coupling and the self force

We consider the evolution of the orbit of a spinning compact object in a quasi-circular, planar orbit around a Schwarzschild black hole in the extreme mass ratio limit. We compare the contributions to the orbital evolution of both spin-orbit coupling and the local self force. Making assumptions on the behavior of the forces, we suggest that the decay of the orbit is dominated by radiation reaction, and that the conservative effect is typically dominated by the spin force. We propose that a reasonable approximation for the gravitational waveform can be obtained by ignoring the local self force, for adjusted values of the parameters of the system. We argue that this approximation will only introduce small errors in the astronomical determination of these parameters.Comment: 11 pages, 7 figure

Temporal and dimensional effects in evolutionary graph theory

The spread in time of a mutation through a population is studied analytically and computationally in fully-connected networks and on spatial lattices. The time, t_*, for a favourable mutation to dominate scales with population size N as N^{(D+1)/D} in D-dimensional hypercubic lattices and as N ln N in fully-connected graphs. It is shown that the surface of the interface between mutants and non-mutants is crucial in predicting the dynamics of the system. Network topology has a significant effect on the equilibrium fitness of a simple population model incorporating multiple mutations and sexual reproduction. Includes supplementary information.Comment: 6 pages, 4 figures Replaced after final round of peer revie

SORLA-mediated trafficking of TrkB enhances the response of neurons to BDNF

Stimulation of neurons with brain-derived neurotrophic factor (BDNF) results in robust induction of SORLA, an intracellular sorting receptor of the VPS10P domain receptor gene family. However, the relevance of SORLA for BDNF-induced neuronal responses has not previously been investigated. We now demonstrate that SORLA is a sorting factor for the tropomyosin-related kinase receptor B (TrkB) that facilitates trafficking of this BDNF receptor between synaptic plasma membranes, post-synaptic densities, and cell soma, a step critical for neuronal signal transduction. Loss of SORLA expression results in impaired neuritic transport of TrkB and in blunted response to BDNF in primary neurons; and it aggravates neuromotoric deficits caused by low BDNF activity in a mouse model of Huntington's disease. Thus, our studies revealed a key role for SORLA in mediating BDNF trophic signaling by regulating the intracellular location of TrkB

An aperture masking mode for the MICADO instrument

MICADO is a near-IR camera for the Europea ELT, featuring an extended field (75" diameter) for imaging, and also spectrographic and high contrast imaging capabilities. It has been chosen by ESO as one of the two first-light instruments. Although it is ultimately aimed at being fed by the MCAO module called MAORY, MICADO will come with an internal SCAO system that will be complementary to it and will deliver a high performance on axis correction, suitable for coronagraphic and pupil masking applications. The basis of the pupil masking approach is to ensure the stability of the optical transfer function, even in the case of residual errors after AO correction (due to non common path errors and quasi-static aberrations). Preliminary designs of pupil masks are presented. Trade-offs and technical choices, especially regarding redundancy and pupil tracking, are explained.Comment: SPIE 2014 Proceeding -- Montrea

A Population Genetic Approach to the Quasispecies Model

A population genetics formulation of Eigen's molecular quasispecies model is proposed and several simple replication landscapes are investigated analytically. Our results show a remarcable similarity to those obtained with the original kinetics formulation of the quasispecies model. However, due to the simplicity of our approach, the space of the parameters that define the model can be explored. In particular, for the simgle-sharp-peak landscape our analysis yelds some interesting predictions such as the existence of a maximum peak height and a mini- mum molecule length for the onset of the error threshold transition.Comment: 16 pages, 4 Postscript figures. Submited to Phy. Rev.