Enhancing native chemical ligation for challenging chemical protein syntheses

Native chemical ligation has enabled the chemical synthesis of proteins for a wide variety of applications (e.g., mirror-image proteins). However, inefficiencies of this chemoselective ligation in the context of large or otherwise challenging protein targets can limit the practical scope of chemical protein synthesis. In this review, we focus on recent developments aimed at enhancing and expanding native chemical ligation for challenging protein syntheses. Chemical auxiliaries, use of selenium chemistry, and templating all enable ligations at otherwise suboptimal junctions. The continuing development of these tools is making the chemical synthesis of large proteins increasingly accessible.     

Leaching requirement conceptual models for reactive salt

Accurate prediction of the leaching requirements (Lr) of crops and striving to attain them is essential for efficient irrigation water use. Solute modeling was extended to develop four Lr conceptual models that do not neglect solute reactions in the root-zone, surface evaporation, and the influence of immobile wetted pore space. The models were based on: (i) the water movement equation which included an exponential water-uptake function (-e) or the 40-30-20-10 water-uptake function (-4); (ii) the solute movement equation for a reactive salt of a linear reaction term (the Lrchem-e and Lrchem-4 models); or the employment of output (salinity of soil solution, EC vs concentration factor, CF) of the SAO comprehensive chemical model (the LrSAO-e and LrSAO-4 models); and (iii) the inclusion of an effective soil solution volume in the transport equations. The root-zone average relative effective soil solution volume νeff (L | L50, p) was of sigmoidal response to leaching fraction (L) with two adjustable parameters L50 and p; the root-zone average reduced retention coefficient decreased linearly with L; and salt concentration at soil surface was related to salt concentration of irrigation water (ECi) by the fraction of irrigation water that evaporated (∈). The resulted concentration profiles indicated the salt behaved as a conservative one down to a threshold depth (xs) below of which salt was retained and precipitated. The depth of the conservative-salt front, xs increased with L and the 40-30-20-10 water-uptake pattern overestimated the xs depth relative to the exponential pattern. Concentration profiles were integrated to compute the root-zone average salinity, which was converted to crop salt-tolerance threshold (AE). The four conceptual models were successfully calibrated using experimental AE/ECi vs. Lr data with the input parameter values: ς = 0.27, p = 1.44, L50 = 0.16, ω = 2, and ∈ = 0 or 0.1 for the exponential or the 40-30-20-10 pattern, respectively; where ς is relative root length parameter and ω is a weighing parameter. No significant difference existed between the four model correlations at the 0.05 level. The four models require ECi and AE of the crop as input for Lr prediction. Sensitivity analysis revealed predicted Lr was sensitive the least to error in ∈. For tolerant and moderately tolerant crops Lr was sensitive the most to ς, and for sensitive crops to L50 and p. Model verification and validation were discussed. In deriving the present Lr models, no osmotic adjustment was required and both the exponential and the 40-30-20-10 water uptake patterns were, equivalently, applicable. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of Asp297 of the AT2 receptor in high-affinity binding to different peptide ligands

To determine how ligand-receptor interaction is affected by the charges of the amino acids at position 2 of the ligands and position 297 of the AT2 receptor, we generated the Asp297Lys mutant of AT2 and a ligand SarAsp²Ile. Asp297Lys mutant lost affinity to Ang II and SarIle however retained partial affinity to ¹²⁵I-CGP42112A. The SarAsp²Ile had high affinity to Asp297Lys (IC₅₀3.5nM) and partial affinity to the AT2 (IC₅₀15nM). Therefore, not only the charge, but also the length of the side arms of the amino acids at position 2 of the ligand and position 297 of the receptor affect their interaction.     

Gas exchange and chlorophyll fluorescence responses of three south-western Yucca species to elevated CO2 and high temperature

The ability of seedlings to tolerate temperature extremes is important in determining the distribution of perennial plants in the arid south-western USA, and the manner in which elevated CO₂ impacts the ability of plants to tolerate high temperatures is relatively unknown. Whereas the effects of chronic high temperature (30–38°C) and elevated CO₂ are comparatively well understood, little research has assessed plant performance in elevated CO₂ during extreme (> 45 °C) temperature events. We exposed three species of Yucca to 360 and 700 μmol CO₂ mol^–1 for 8 months, then 9 d of high temperature (up to 53 °C) to evaluate the impacts of elevated CO₂ on the potential for photosynthetic function during external high temperature. Seedlings of a coastal C₃ species (Yucca whipplei), a desert C₃ species (Yucca brevifolia), and a desert CAM species (Yucca schidigera), were used to test for differences among functional groups. In general, Yuccas exposed to elevated CO₂ showed decreases in carboxylation efficiency as compared with plants grown at ambient before the initiation of high temperature. The coastal species (Y. whipplei) showed significant reductions (33%) in CO₂ saturated maximum assimilation rate (A_max), but the desert species (Y. brevifolia and Y. schidigera) showed no such reductions in A_max. Stomatal conductance was lower in elevated CO₂ as compared with ambient throughout the temperature event; however, there were species-specific differences over time. Elevated CO₂ enhanced photosynthesis in Y. whipplei at high temperatures for a period of 4 d, but not for Y. brevifolia or Y. schidigera. Elevated CO₂ offset photoinhibition (measured as F_v/F_m) in Y. whipplei as compared with ambient CO₂, depending on exposure time to high temperature. Stable F_v/F_m in Y. whipplei occurred in parallel with increases in the quantum yield of photosystem II (ΦPSII) at high temperatures in elevated CO₂. The value of ΦPSII remained constant or decreased with increasing temperature in all other treatment and species combinations. This suggests that the reductions in F_v/F_m resulted from thermal energy dissipation in the pigment bed for Y. brevifolia and Y. schidigera. The greater efficiency of photosystem II in Y. whipplei helped to maintain photosynthetic function at high temperatures in elevated CO₂. These patterns are in contrast to the hypothesis that high temperatures in elevated CO₂ would increase the potential for photoinhibition. Our results suggest that elevated CO₂ may offset high-temperature stress in coastal Yucca, but not in those species native to drier systems. Therefore, in the case of Y. whipplei, elevated CO₂ may allow plants to survive extreme temperature events, potentially relaxing the effects of high temperature on the establishment in novel habitats.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers

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Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19

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