Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or ERAD. Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10 and the Asi complex, which resides at the nuclear envelope (NE). These ER/NE membrane-associated RING-type E3 ligases recognize and ubiquitylate defective proteins in cooperation with the E2 conjugating enzyme Ubc7 and the obligatory Ubc7 co-factor Cue1. Interaction of Ubc7 with the RING domains of its cognate E3 Ub-protein ligases stimulates the formation of isopeptide (amide) Ub-Ub linkages. Each isopeptide bond is formed by transfer of a Ubc7-linked activated Ub to a lysine side chain of an acceptor Ub. Multiple Ub transfer reactions form a poly-Ub chain that targets the conjugated protein for degradation by the proteasome. To study the mechanism of Ub-Ub bond formation, this reaction is reconstituted in a cell-free system consisting of recombinant E1, Ub, Ubc7, its co-factor Cue1, and the RING domain of either Doa10 or Hrd1. Here we provide detailed protocols for the purification of the required recombinant proteins and for the reactions that produce an Ub-Ub bond, specifically, the formation of a Ubc7~Ub thiolester (Ub charging) and subsequent formation of the isopeptide Ub-Ub linkage (Ub transfer). These protocols also provide a useful guideline for similar in vitro ubiquitylation reactions intended to explore the mechanism of other Ub-conjugation systems.
Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions recurrently rearranged in cancer. Many CFSs are enriched with AT-dinucleotide rich sequences (AT-DRSs) which have the potential to form stable secondary structures upon unwinding the double helix during DNA replication. These stable structures can potentially perturb DNA replication progression, leading to genomic instability. Using site-specific targeting system, we show that targeted integration of a 3.4 kb AT-DRS derived from the human CFS FRA16C into a chromosomally stable region within the human genome is able to drive fragile site formation under conditions of replication stress. Analysis of >1300 X chromosomes integrated with the 3.4 kb AT-DRS revealed recurrent gaps and breaks at the integration site. DNA sequences derived from the integrated AT-DRS showed in vitro a significantly increased tendency to fold into branched secondary structures, supporting the predicted mechanism of instability. Our findings clearly indicate that intrinsic DNA features, such as complexed repeated sequence motifs, predispose the human genome to chromosomal instability.
Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far.
Shahar Sukenik, Daniel Harries, and Assaf Friedler. 2019. “Biophysical Chemistry.” In Elsevier Reference Module in Chemistry, Molecular Sciences and Chemical Engineering. Waltham, MA: Elsevier. Publisher's VersionAbstract
Biophysical chemistry is a branch of the multidisciplinary study of biophysics. The field is devoted to a quantitative analysis of biological systems using experimental, theoretical, and computational tools. In contrast to a physics-centered approach for biophysics that deals with forces and scaling laws, or a biology-centered view that deals with the phenotype of the studied system, biophysical chemistry focuses on the molecular level. Unlike biochemistry, which often focuses on chemical reactions driving biological systems, biophysical chemistry is aimed at the collection and analysis of quantitative data to provide predictive physical models describing biological phenomena occurring at the molecular level. Biophysical chemistry aims to bridge the physical and biological disciplines: in biological systems physical forces and interactions are mediated through molecules, which ultimately determine phenotype. The experimental and theoretical tools of biophysical chemistry have demonstrated significant success in unraveling multiple basic molecular mechanisms that govern biological processes. Here we highlight some of the important contemporary areas and questions currently studied in the field of biophysical chemistry. Since molecules are at the center of this study, we focus our discussion on three classes of molecules essential for all living organisms: proteins, nucleic acids, and lipids.
Size polymorphism is common in bees, and is determined by environmental factors such as temperature, brood cell size, and the diet provided to developing larvae. In social bees, these factors are further influenced by intricate interactions between the queen, workers, and the developing brood which eventually determine the final size and caste of developing larvae. Environmental and social factors act in part on juvenile hormone and ecdysteroids, which are key hormonal regulators of body size and caste determination. In some social bees, body size variation is central for social organization because it structures reproductive division of labor, task allocation among workers, or both. At ecological scales, body size also impacts bee-mediated pollination services in solitary and social species by influencing floral visitation and pollination efficacy.
Sleep is ubiquitous in vertebrates and invertebrates and its loss is typically associated with reduced performance, health, or survival, for reasons that are yet unclear [1—3]. Nevertheless, some animals can reduce sleep for increasing foraging time [4], under predation risk [5—8], during seasonal migration [9—11], or for having greater mating opportunities [12,13]. Here we tested the hypothesis that social bumble bee (Bombus terrestris) workers give-up sleep for improving brood-care. We combined video- recordings, detailed behavioral analyses, sleep-deprivation experiments, and response-threshold assessments, to characterize the sleep behavior of worker bees and showed that immobility bouts of ≥ 5' provide a reliable proxy for sleep. We next used this index to study sleep with an automated video-based activity monitoring system. We found that isolated workers severely reduce sleep time in the presence of both larvae that need to be fed, or pupae that do not. Reduced sleep was also correlated with around-the-clock activity and wax-pot building, which are typical for nest-founding mother queens. Cocoons, from which we removed the pupae, elicited a similar but transient sleep-loss in tending workers, suggesting that the pupa effect on sleep is mediated by pheromonal signals. Sleep time increased following brood removal, but remained lower compared to control bees, suggesting that the brood modulated sleep-need. This first evidence for brood modulation of sleep in an insect suggests that plasticity in sleep can evolve as a mechanism to improve care for dependent juveniles, even in social insect workers that do not care for their own offspring.
Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.
Our Cr:ZnSe laser amplifier, seeded by parametric difference mixing, produces 72fs long pulses at the central wavelength of 2.37&\#x00B5;m. The stability of the carrier-to-envelope phase of the amplified seed pulses, attained at the stage of their parametric generation, is preserved through 6 orders of magnitude of laser amplification.
