Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry—a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel—such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

The TIM barrel fold is among the most versatile enzyme scaffolds in nature (1, 2). It consists of eight parallel β-strands surrounded by eight α-helices in a (β/α)₈ topology. The parallel β-strands confer a polarity to the domain—a “top” and “bottom.” Enzyme active sites in nature are usually located at the center of the top (3), where side chains from all eight strands are directed toward the center of the barrel. Additional substrate-binding components are typically formed from irregular, extended loops that connect the strands and helices (4). Below the active site, an extensive hydrophobic core between the β-barrel and the α-helices provides considerable stability, allowing TIM barrel active sites to diversify with minimal effects on overall fold stability (5).The engineering of natural TIM barrels, both by directed evolution and design, has resulted in enzymes with altered substrate specificities, enhanced reaction rates, and even new-to-nature chemistries (6–13). The de novo design of catalytic functions, however, has been challenged in part by the difficulty in programming flexible loops; the most successful approaches to date have reused loops found in nature (14, 15). An artificial TIM barrel designed with atomic-level accuracy was created previously using the Rosetta macromolecular and design platform (16, 17). This protein (sTIM11), which has no native function and no natural sequence homologs, was created by repeating four copies of a 46-amino acid (β/α)₂ unit about a central symmetry axis. It was expressed as a single chain, but the internal symmetry left open the possibility of converting the barrel into a homotetramer or homodimer by expressing one or two 46-residue repeats, then allowing these parts to self-assemble in solution to form a complete TIM barrel. More recently, this scaffold has been reengineered to improve thermodynamic and kinetic stability, yielding a series of improved variants dubbed DeNovoTIMs (18). De novo protein design is inspired by but not limited to nature’s solutions. To generate a protein scaffold that might support the incorporation of catalytic cofactors, we aimed for an alternative to loop design: fusing ordered protein domains directly on top of a DeNovoTIM variant.Many natural proteins require a metal ion to carry out their function. Such metalloproteins are involved in numerous biological processes, including metal ion storage and transport, sensing and signaling, electron transfer pathways, and catalysis. Computationally designed proteins may serve as a starting point to engineer artificial metalloproteins with specific binding sites for a metal ion of choice. Thus, in addition to engineering a suitable lid atop DeNovoTIM, we set out to introduce a de novo metal-binding site for large, trivalent lanthanide cations, which possess unique electronic and photophysical properties (19). The f-block elements exhibit long-lived luminescence, which can be enhanced through energy transfer from a close-by antenna molecule, an approach that is exploited in so-called lanthanide-binding tags (20, 21). De novo designed peptides were shown previously to form lanthanide-binding coiled coils with potential applications as paramagnetic MRI contrast agents (22, 23). Their strong Lewis acidity also makes lanthanides powerful catalysts for various synthetically useful reactions (24), although their biological relevance was only recently discovered, when lanthanide-dependent methanol dehydrogenases were isolated from certain methylotrophic and methanotrophic bacteria (25, 26). Here, we introduce a de novo lanthanide-binding protein with ultra-high affinity, which provides a starting point for the future engineering of lanthanide-dependent biotechnological tools and artificial enzymes.     

Influence of sleep disturbance on quality of life of patients with epilepsy.

The frequency of sleep disturbances in patients with epilepsy and their impact on quality of life (QoL) have been documented in a few reports, and the results are conflicting. We identified 124 consecutive epilepsy out-patients who visited the epilepsy out-patient clinics at the University Hospital of Alexandroupolis, the AHEPA Hospital in Thessaloniki and the Aeginitio Hospital in Athens. We measured excessive daytime sleepiness (EDS) with the Epworth Sleepiness Scale (ESS), obstructive sleep apnea (OSA) with the Sleep Apnea scale of the Sleep Disorders Questionnaire (SA-SDQ), and insomnia with the Athens Insomnia Scale (AIS). We evaluated quality of life by the Quality of Life in Epilepsy Inventory (QOLIE-31). EDS was found in 16.9% (21/124) of epileptic patients, OSA in 28.2% (35/124), and insomnia in 24.6% (30/122). In multivariate analysis, we found that insomnia was an independent negative factor for Total score (p<0.001), Overall QoL (p=0.002), Emotional well-being (p<0.001), Energy/fatigue (p<0.001), Cognitive functioning (p=0.04) and Social functioning (p=0.03), and OSA only for Cognitive functioning (p=0.01). According to our findings, EDS, OSA, and insomnia are frequent in epileptic patients. Epileptic patients with sleep disturbance, mainly insomnia, have significant QoL impairment.     

Aripiprazole in the acute treatment of male patients with schizophrenia: effectiveness, acceptability, and risks in the inner-city hospital setting

Aripiprazole, a novel atypical antipsychotic that acts as a partial agonist at the dopamine D₂ receptors, has been reported to be effective in the treatment of chronic schizophrenia. However, the risks and benefits of using aripiprazole in the acute hospital setting to treat severe psychotic disorders are unclear. This naturalistic study assessed the effectiveness of aripiprazole monotherapy in a group of actively psychotic male patients (n = 10) with schizophrenia who were admitted to an inner-city acute psychiatric unit. Most patients (n = 7) responded to aripiprazole treatment, which was well tolerated and significantly ameliorated psychotic symptoms after 2–3 weeks. Patients who responded to it could be safely discharged on aripiprazole monotherapy. Side effects observed were mostly mild and transient, and included extrapyramidal symptoms (n = 1) and neutropenia (n = 1). Aripiprazole also remarkably attenuated dyskinetic movements in 1 patient with severe tardive dyskinesia, thereby suggesting that it may be useful in the treatment of other disorders that are also associated with dopamine dysfunction. Results showed that aripiprazole can be safely and effectively employed in the hospital setting to treat severely psychotic patients with schizophrenia, but further studies are required to establish the full range of adverse reactions and therapeutic indications associated with its use.     

Carotid plaque echomorphology and serum vascular endothelial growth factor levels

Neovascularization in atherosclerotic plaques plays an essential role in the progression and rupture of plaques. Vascular endothelial growth factor (VEGF) is an important angiogenic factor. Echomorphologic evaluation of carotid plaques using computer-assisted imaging was found to have a good correlation with the histology of the lesion. The aim of this study was to investigate whether the serum VEGF level could be a determinant of the echomorphology of the carotid plaque. In 28 carotid plaques causing 60-99% stenosis, serum VEGF levels and the mean gray value (MGV) of three-dimensional image of the carotid plaques were measured. A statistically significant inverse correlation was found between serum VEGF concentrations and MGVs (Spearman's correlation coefficient: -0.415, p = 0.028). Our finding indicates that in patients with > or =60% carotid stenosis the serum VEGF levels are associated with the echogenicity of the atherosclerotic plaque.     

Bilateral central retinal artery occlusions in a patient with activated protein C resistance

We describe the case of a 42-year-old woman who had two episodes of visual loss, first in one and later in the other eye, which caused bilateral reduction of visual acuity to 2/10 with optic atrophy. Detailed clinical and laboratory assessment revealed an activated protein C (APC) resistance and no other obvious cause. APC resistance has not been previously reported as an etiologic factor of central retinal artery occlusion.