Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria.

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, implement combinatorial regulation, and reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

Kinetic FRET Assay to Measure Binding-Induced Conformational Changes of Nucleic Acids.

The interaction of small molecules or proteins with RNA or DNA often involves changes in the nucleic acid (NA) folding and structure. A biophysical characterization of these processes helps us to understand the underlying molecular mechanisms. Here, we propose kinFRET (kinetics Förster resonance energy transfer), a real-time ensemble FRET methodology to measure binding and folding kinetics. With kinFRET, the kinetics of conformational changes of NAs (DNA or RNA) upon analyte binding can be directly followed via a FRET signal using a chip-based biosensor. We demonstrate the utility of this approach with two representative examples. First, we monitored the conformational changes of different formats of an aptamer (MN19) upon interaction with small-molecule analytes. Second, we characterized the binding kinetics of RNA recognition by tandem K homology (KH) domains of the human insulin-like growth factor II mRNA-binding protein 3 (IMP3), which reveals distinct kinetic contributions of the two KH domains. Our data demonstrate that kinFRET is well suited to study the kinetics and conformational changes of NA-analyte interactions.

Protein photodegradation in the visible range? Insights into protein photooxidation with respect to protein concentration.

Visible light (400-800 nm) can lead to photooxidation of protein formulations, which might impair protein integrity. However, the relevant mechanism of photooxidation upon visible light exposure is still unclear for therapeutic proteins, since proteinogenic structures do not absorb light in the visible range. Here, we show that exposure of monoclonal antibody formulations to visible light, lead to the formation of reactive oxygen species (ROS), which subsequently induce specific protein degradations. The formation of ROS and singlet oxygen upon visible light exposure is investigated using electron paramagnetic resonance (EPR) spectroscopy. We describe the initial formation of ROS, most likely after direct reaction of molecular oxygen with a triplet state photosensitizer, generated from intersystem crossing of the excited singlet state. Since these radicals affect the oxygen content in the headspace of the vial, we monitored photooxidation of these mAb formulations. With increasing protein concentrations, we found (i) a decreasing headspace oxygen content in the sample, (ii) a higher relative number of radicals in solution and (iii) a higher protein degradation. Thus, the protein concentration dependence indicates the presence of higher concentration of a currently unknown photosensitizer.

Investigating photodegradation of antibodies governed by the light dosage.

The present study investigated the photodegradation of three different monoclonal antibodies (mAb) by visible light. Several chromatographic techniques, such as size-exclusion and hydrophobic interaction chromatography as well as mass spectrometry were used to measure relative changes of various oxidation related monoclonal antibody species. The results show that visible light is indeed capable of inducing the formation of protein photo-oxidation products, such as acidic, basic, hydrophilic, and several other protein species with altered physicochemical properties. Although, the formation rate of degradants of these three protein species was dependent on the light source's intensity (I), their yield is clearly correlated to the applied light dosage (ld), which is defined as the product of light intensity I and irradiation time t (light dosage = I·t). Hence, our findings indicate that the degradation of monoclonal antibodies can be described according to the Bunsen-Roscoe reciprocity law. This correlation can be useful to assess the impact of photodegradation of biologics with regards to changes in light intensity and/or duration of light exposure of the protein, e.g. during the manufacturing of biologics.

Photo-Oxidation of Therapeutic Protein Formulations: From Radical Formation to Analytical Techniques.

UV and ambient light-induced modifications and related degradation of therapeutic proteins are observed during manufacturing and storage. Therefore, to ensure product quality, protein formulations need to be analyzed with respect to photo-degradation processes and eventually protected from light exposure. This task usually demands the application and combination of various analytical methods. This review addresses analytical aspects of investigating photo-oxidation products and related mediators such as reactive oxygen species generated via UV and ambient light with well-established and novel techniques.

Nucleotide binding kinetics and conformational change analysis of tissue transglutaminase with switchSENSE.

Function, activity, and interactions of proteins crucially depend on their three-dimensional structure and are often regulated by effector binding and environmental changes. Tissue transglutaminase (Transglutaminase 2, TG2) is a multifunctional protein, allosterically regulated by nucleotides and Ca2+ ions, which trigger opposing conformational changes. Here we introduce switchSENSE as a versatile tool for TG2 characterization and provide novel insights into protein conformation as well as analyte binding kinetics. For the first time, we succeeded in measuring the kinetic rate constants and affinities (kon, koff, KD) for guanosine nucleotides (GMP, GDP, GTP, GTPγS). Further, the conformational changes induced by GDP, Ca2+ and the covalent inhibitor Z-DON were observed by changes in TG2's hydrodynamic diameter. We confirmed the well-known compaction by guanosine nucleotides and extension by Ca2+, and provide evidence for TG2 conformations so far not described by structural analysis. Moreover, we analyze the influence of the peptidic Z-DON inhibitor and the R580A mutation on the conformational responsiveness of TG2 to its natural effectors. In summary, this work shows how the combination of structural and kinetic information obtained by switchSENSE opens new perspectives for the characterization of conformationally active proteins and their interactions with ligands, e.g. potential drug candidates.

Electrical Actuation of a DNA Origami Nanolever on an Electrode.

Development of electrically powered DNA origami nanomachines requires effective means to actuate moving origami parts by externally applied electric fields. We demonstrate how origami nanolevers on an electrode can be manipulated (switched) at high frequency by alternating voltages. Orientation switching is long-time stable and can be induced by applying low voltages of 200 mV. The mechanical response time of a 100 nm long origami lever to an applied voltage step is less than 100 µs, allowing dynamic control of the induced motion. Moreover, through voltage assisted capture, origamis can be immobilized from folding solution without purification, even in the presence of excess staple strands. The results establish a way for interfacing and controlling DNA origamis with standard electronics, and enable their use as moving parts in electro-mechanical nanodevices.

YbiB from Escherichia coli, the Defining Member of the Novel TrpD2 Family of Prokaryotic DNA-binding Proteins.

We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (KD = 10-100 nM) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.

Protein analysis by time-resolved measurements with an electro-switchable DNA chip.

Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements.

Quantitation of affinity, avidity, and binding kinetics of protein analytes with a dynamically switchable biosurface.

A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers' dynamic motion. We demonstrate the quantitation of binding kinetics (k(on), k(off) rate constants), dissociation constants (K(D) in the pM regime), and the influence of competitive binders (EC(50) values). Moreover, the "switchSENSE" method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA(3)) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins' individual chemical environments significantly influence the His(6)-NTA(3) interaction.

Conformations of end-tethered DNA molecules on gold surfaces: influences of applied electric potential, electrolyte screening, and temperature.

We describe the behavior of 72mer oligonucleotides that are end-tethered to gold surfaces under the influence of applied electric fields. The DNA extension is measured by fluorescence energy transfer as a function of the DNA hybridization state (single- and double-stranded), the concentration of monovalent salt in solution (100 microM to 1 M NaCl), the applied electrode potential (-0.6 to +0.1 V vs Pt), and the temperature (1 to 50 degrees C). At high ionic strength, the DNA conformations are very robust and independent of the applied electrode potential and temperature variations. In solutions of medium ionic strength, the DNA conformation can be manipulated efficiently by applying bias potentials to the Au electrodes. The molecules are repelled at negative potentials and attracted to the surface at positive potentials. The conformation transition occurs abruptly when the electrode bias is swept by merely 0.1 V across the transition potential, which shifts negatively when the salinity is decreased. The behavior can be understood by electrostatic screening arguments and, in the case of single-stranded DNA, when secondary structures are taken into account. At low ionic strength, the experiments reveal an intriguing temperature-dependent stiffening of single-stranded DNA, which can be rationalized by combining counterion condensation theory with the Odjik-Skolnick-Fixman description of the electrostatic persistence length and the unstacking of bases at elevated temperatures.

Detection and size analysis of proteins with switchable DNA layers.

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

Remember judgments and the constraint of direct experience.

The most direct assessment of episodic memory is provided by Remember versus Know judgments of recalled or recognised items. We investigate whether Remember judgments reflect episodic memories as a re-experience of formerly experienced events (mental time travel). If they do, they must obey the direct experience constraint: only directly experienced events can be re-experienced but not when the event is known through indirectly conveyed information. In two Experiments participants saw simple events in Power Point, e.g. a car exploding. In the direct experience condition these events were directly perceived. In three further conditions information about the object (particular car), the kind of event (explosion), or both were verbally conveyed. After controlling for a potential encoding specificity effect in Experiment 1, the frequency of Remember judgments was twice as high in the direct experience condition than in the other three conditions. This suggests that Remember judgments are--at least to some degree--subject to the direct experience constraint.

Do long-term hospitalised patients benefit from discharge into the community?

OBJECTIVE: The study investigated whether long-stay patients would benefit from discharge into the community in Berlin, Germany. METHOD: In a prospective controlled study, all long-term hospitalised psychiatric patients from a defined catchment area were assessed using established standardised instruments. Quality of life, treatment satisfaction, needs and psychopathology were re-assessed in 63 non-discharged patients 1.5 years later, and in 65 resettled patients 1 year after discharge. RESULTS: Discharged patients were younger and had spent less time in psychiatric hospitalisation. Whilst patients who remained in hospital care did not show significant changes over time, discharged patients did. Changes in subjective quality of life and total number of needs - but not in psychopathology, unmet needs, and treatment satisfaction - were significantly more favourable in resettled patients as compared to the control group. CONCLUSION: The findings are in line with other studies and suggest that long-stay patients can benefit from discharge into the community, particularly with respect to their quality of life. Positive changes in the process of deinstitutionalisation seem not dependent on the specific national context, and also apply to younger patients who have not yet spent 10 or more years in psychiatric hospitals.

Subjective evaluation: is there more than one criterion?

Previous cross-sectional investigations have shown that subjective evaluation criteria (criteria that are used in psychiatric research for evaluating care based on patients' statements) do overlap and that there exists a single general factor underlying all these criteria. In this study, we tested longitudinally and in two different samples of schizophrenia patients the distinctness and covariation at baseline and at followup of three common subjective evaluation criteria (subjective quality of life, self-rated needs, and self-reported symptoms). Scores were intercorrelated at both baseline and followup and showed some intercorrelations over time, suggesting temporal covariation. One stable subjective appraisal factor was identified at both baseline and followup, summarizing a negative subjective quality of life and more symptoms and needs. This factor explained 50 percent to 69 percent of the variance. It was found to be strongly associated with observer-rated mood and was mainly predicted by reporting dark thoughts and being dissatisfied with life as a whole. In subjective evaluation, there appears to be a need to distinguish between a general appraisal factor and specific aspects of different criteria. Therefore, future research needs to focus on how the general factor can be assessed more directly and to identify how the specific variance of different criteria independent of that factor can be maximized.