In silico prediction of mutant HIV-1 proteases cleaving a target sequence

HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636 -- 1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidates for specificity and cleavability towards the target sequence

Redox characteristics of the eukaryotic cytosol

AbstractThe eukaryotic cytoplasm has long been regarded as a cellular compartment in which the reduced state of protein cysteines is largely favored. Under normal conditions, the cytosolic low-molecular weight redox buffer, comprising primarily of glutathione, is highly reducing and reactive oxygen species (ROS) and glutathionylated proteins are maintained at very low levels. In the present review, recent progress in the understanding of the cytosolic thiol–disulfide redox metabolism and novel analytical approaches to studying cytosolic redox properties are discussed. We will focus on the yeast model organism, Saccharomyces cerevisiae, where the combination of genetic and biochemical approaches has brought us furthest in understanding the mechanisms underlying cellular redox regulation. It has been shown in yeast that, in addition to the enzyme glutathione reductase, other mechanisms may exist for restricting the cytosolic glutathione redox potential to a relatively narrow interval. Several mutations in genes involved in cellular redox regulation cause ROS accumulation but only moderate decreases in the cytosolic glutathione reducing power. The redox regulation in the cytosol depends not only on multiple cytosolic factors but also on the redox homeostasis of other compartments like the secretory pathway and the mitochondria. Possibly, the cytosol is not just a reducing compartment surrounding organelles with high oxidative activity but also a milieu for regulation of the redox status of more than one compartment. Although much has been learned about redox homeostasis and oxidative stress response several important aspects of the redox regulation in the yeast cytosol are still unexplained

Substitutional landscape of a split fluorescent protein fragment using high-density peptide microarrays

Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide

Armenia: What drives first movers and how can their efforts be scaled up?

The paper examines ways to expand the contribution of the Armenian diaspora to Armenia’s long-term development agenda. It identifies factors that could explain the involvement and dynamics of a small group of entrepreneurs from the diaspora who have been active in and with Armenia. Based on these findings, it develops recommendations, consistent with the diaspora’s institutional capabilities, for increasing the number of such business activists and transforming diaspora efforts from humanitarian relief campaigns to business initiatives and development projects. The findings are based on detailed interviews with a group of prominent diaspora activists

Computational redesign of thioredoxin is hypersensitive towards minor conformational changes in the backbone template

Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations

Age determination of galaxy merger remnant stars using asteroseismology

The Milky Way was shaped by the mergers with several galaxies in the past. We search for remnant stars that were born in these foreign galaxies and assess their ages in an effort to put upper limits on the merger times and thereby better understand the evolutionary history of our Galaxy. Using 5D-phase space information from Gaia eDR3, radial velocities from Gaia DR2 and chemical information from apogee DR16, we kinematically and chemically select 21 red giant stars belonging to former dwarf galaxies that merged with the Milky Way. With added asteroseismology from Kepler and K2, we determine the ages of the 21 ex situ stars and 49 in situ stars with an average σage/age of ∼31 per cent. We find that all the ex situ stars are consistent with being older than 8 Gyr. While it is not possible to associate all the stars with a specific dwarf galaxy, we classify eight of them as Gaia-Enceladus/Sausage stars, which is one of the most massive mergers in our Galaxy's history. We determine their mean age to be 9.5 ± 1.3 Gyr consistent with a merger time of 8-10 Gyr ago. The rest of the stars are possibly associated with Kraken, Thamnos, Sequoia, or another extragalactic progenitor. The age determination of ex situ stars paves the way to more accurately pinning down when the merger events occurred and hence provide tight constraints useful for simulating how these events unfolded.Funding for the Stellar Astrophysics Centre was provided by The Danish National Research Foundation (grant agreement no. DNRF106). AH acknowledges support from a Spinoza prize from the Netherlands Research Council (NWO). HHK gratefully acknowledges financial support from a Fellowship at the Institute for Advanced Study. AS acknowledges support from the European Research Council Consolidator Grant funding scheme (project ASTEROCHRONOMETRY, G.A. n. 772293, http://www.asterochronometry.eu). JMDK gratefully acknowledges funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through an Emmy Noether Research Group (grant number KR4801/1-1), as well as from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme via the ERC Starting Grant MUSTANG (grant agreement number 714907). CL acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 852839). JY acknowledges partial support from ERC Synergy Grant WHOLE SUN 810218

Asteroseismology and Spectropolarimetry of the Exoplanet Host Star λ Serpentis

The bright star lambda Ser hosts a hot Neptune with a minimum mass of 13.6 M & OPLUS; and a 15.5 day orbit. It also appears to be a solar analog, with a mean rotation period of 25.8 days and surface differential rotation very similar to the Sun. We aim to characterize the fundamental properties of this system and constrain the evolutionary pathway that led to its present configuration. We detect solar-like oscillations in time series photometry from the Transiting Exoplanet Survey Satellite, and we derive precise asteroseismic properties from detailed modeling. We obtain new spectropolarimetric data, and we use them to reconstruct the large-scale magnetic field morphology. We reanalyze the complete time series of chromospheric activity measurements from the Mount Wilson Observatory, and we present new X-ray and ultraviolet observations from the Chandra and Hubble space telescopes. Finally, we use the updated observational constraints to assess the rotational history of the star and estimate the wind braking torque. We conclude that the remaining uncertainty on the stellar age currently prevents an unambiguous interpretation of the properties of lambda Ser, and that the rate of angular momentum loss appears to be higher than for other stars with a similar Rossby number. Future asteroseismic observations may help to improve the precision of the stellar age