High-yield Escherichia coli -based cell-free expression of human proteins

Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [15N,1H]-HSQC NM

Automatically identifying eviction cases and outcomes within case law of Dutch Courts of First Instance

In this paper we attempt to identify eviction judgements within all case law published by Dutch courts in order to automate data collection, previously conducted manually. To do so we performed two experiments. The first focused on identifying judgements related to eviction, while the second focused on identifying the outcome of the cases in the judgements (eviction vs. dismissal of the landlord’s claim). In the process of conducting the experiments for this study, we have created a manually annotated dataset with eviction-related judgements and their outcomes

Removing Barriers, Integrating Research, Spreading Excellence: The European Satellite Communications Network of Excellence "SatNEx"

Within the recently launched 6th Research Framework Programme of the European Commission, 21 major players in satellite communications research have joined forces to implement the European Satellite Communications Network of Excellence (SatNEx). The primary goal of SatNEx is to achieve long-lasting integration of the European research in satellite communication and to develop a common base of knowledge, thus contributing to the realization of the European Research Area. This paper discusses the background and motivation for implementation of the network and highlights the SatNEx mission and key objectives. A top-level overview is then provided including a description of the consortium, the Joint Programme of Activities (JPA) and the time schedule with deliverables and milestones. Finally, an update of ongoing work is presented

Peptides in BioNMR Research

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ?-TIA to the human G-protein coupled ?1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics

Molecular basis for the wide range of affinity found in Csr/Rsm protein-RNA recognition

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protei

Peptide binding affinity redistributes preassembled repeat protein fragments

Designed armadillo repeat proteins (dArmRPs) are modular peptide binders composed of N- and C-terminal capping repeats Y and A and a variable number of internal modules M that each specifically recognize two amino acids of the target peptide. Complementary fragments of dArmRPs obtained by splitting the protein between helices H1 and H2 of an internal module show conditional and specific assembly only in presence of a target peptide (Michel et al., 2018). Here, we investigate dArmRP fragments that already spontaneously assemble with high affinity, e.g. those obtained from splits between entire modules or between helices H2 and H3. We find that the interaction of the peptide with the assembled fragments induces distal conformational rearrangements that suggest an induced fit on a global protein level. A population analysis of an equimolar mixture of an N-terminal and three C-terminal fragments with various affinities for the target peptide revealed predominant assembly of the weakest peptide binder. However, adding target peptide to this mixture altered the population of the protein complexes such that the combination with the highest affinity for the peptide increased and becomes predominant when adding access of peptide, highlighting the feasibility of peptide-induced enrichment of best binders from inter-modular fragment mixtures

Unexpected dynamics in femtomolar complexes of binding proteins with peptides

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders

Analytic Theory of Titans Schumann Resonance: Constraints on Ionospheric Conductivity and Buried Water Ocean

This study presents an approximate model for the atypical Schumann resonance in Titan's atmosphere that accounts for the observations of electromagnetic waves and the measurements of atmospheric conductivity performed with the Huygens Atmospheric Structure and Permittivity, Wave and Altimetry (HASI-PWA) instrumentation during the descent of the Huygens Probe through Titan's atmosphere in January 2005. After many years of thorough analyses of the collected data, several arguments enable us to claim that the Extremely Low Frequency (ELF) wave observed at around 36 Hz displays all the characteristics of the second harmonic of a Schumann resonance. On Earth, this phenomenon is well known to be triggered by lightning activity. Given the lack of evidence of any thunderstorm activity on Titan, we proposed in early works a model based on an alternative powering mechanism involving the electric current sheets induced in Titan's ionosphere by the Saturn's magnetospheric plasma flow. The present study is a further step in improving the initial model and corroborating our preliminary assessments. We first develop an analytic theory of the guided modes that appear to be the most suitable for sustaining Schumann resonances in Titan's atmosphere. We then introduce the characteristics of the Huygens electric field measurements in the equations, in order to constrain the physical parameters of the resonating cavity. The latter is assumed to be made of different structures distributed between an upper boundary, presumably made of a succession of thin ionized layers of stratospheric aerosols spread up to 150 km and a lower quasi-perfect conductive surface hidden beneath the non-conductive ground. The inner reflecting boundary is proposed to be a buried water-ammonia ocean lying at a likely depth of 55-80 km below a dielectric icy crust. Such estimate is found to comply with models suggesting that the internal heat could be transferred upwards by thermal conduction of the crust, while convective processes cannot be ruled out

Long-term soil warming decreases microbial phosphorus utilization by increasing abiotic phosphorus sorption and phosphorus losses

Phosphorus (P) is an essential and often limiting element that could play a crucial role in terrestrial ecosystem responses to climate warming. However, it has yet remained unclear how different P cycling processes are affected by warming. Here we investigate the response of soil P pools and P cycling processes in a mountain forest after 14 years of soil warming (+4 °C). Long-term warming decreased soil total P pools, likely due to higher outputs of P from soils by increasing net plant P uptake and downward transportation of colloidal and particulate P. Warming increased the sorption strength to more recalcitrant soil P fractions (absorbed to iron oxyhydroxides and clays), thereby further reducing bioavailable P in soil solution. As a response, soil microbes enhanced the production of acid phosphatase, though this was not sufficient to avoid decreases of soil bioavailable P and microbial biomass P (and biotic phosphate immobilization). This study therefore highlights how long-term soil warming triggers changes in biotic and abiotic soil P pools and processes, which can potentially aggravate the P constraints of the trees and soil microbes and thereby negatively affect the C sequestration potential of these forests