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Huffman Weber posted an update 6 months, 1 week ago
New enzyme catalysts are usually engineered by repurposing the active sites of natural proteins. Here we show that design and directed evolution can be used to transform a non-natural, functionally naive zinc-binding protein into a highly active catalyst for an abiological hetero-Diels-Alder reaction. The artificial metalloenzyme achieves >104 turnovers per active site, exerts absolute control over reaction pathway and product stereochemistry, and displays a catalytic proficiency (1/KTS = 2.9 × 1010 M-1) that exceeds all previously characterized Diels-Alderases. These properties capitalize on effective Lewis acid catalysis, a chemical strategy for accelerating Diels-Alder reactions common in the laboratory but so far unknown in nature. Extension of this approach to other metal ions and other de novo scaffolds may propel the design field in exciting new directions.Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute, even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. Selleckchem Akt inhibitor To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).Tackling antibiotic resistance necessitates deep understanding of how resource competition within and between species modulates the fitness of resistant microbes. Recent advances in ecological coexistence theory offer a powerful framework to probe the mechanisms regulating intra- and interspecific competition, but the significance of this body of theory to the problem of antibiotic resistance has been largely overlooked. In this Perspective, we draw on emerging ecological theory to illustrate how changes in resource niche overlap can be equally important as changes in competitive ability for understanding costs of resistance and the persistence of resistant pathogens in microbial communities. We then show how different temporal patterns of resource and antibiotic supply, alongside trade-offs in competitive ability at high and low resource concentrations, can have diametrically opposing consequences for the coexistence and exclusion of resistant and susceptible strains. These insights highlight numerous opportunities for innovative experimental and theoretical research into the ecological dimensions of antibiotic resistance.Disturbance and habitat modification by humans can alter animal movement, leading to negative impacts on fitness, survival and population viability. However, the ubiquity and nature of these impacts across diverse taxa has not been quantified. We compiled 208 studies on 167 species from terrestrial and aquatic ecosystems across the globe to assess how human disturbance influences animal movement. We show that disturbance by humans has widespread impacts on the movements of birds, mammals, reptiles, amphibians, fish and arthropods. More than two-thirds of 719 cases represented a change in movement of 20% or more, with increases in movement averaging 70% and decreases -37%. Disturbance from human activities, such as recreation and hunting, had stronger impacts on animal movement than habitat modification, such as logging and agriculture. Our results point to a global restructuring of animal movement and emphasize the need to reduce the negative impacts of humans on animal movement.Haplotype-resolved de novo assembly is the ultimate solution to the study of sequence variations in a genome. However, existing algorithms either collapse heterozygous alleles into one consensus copy or fail to cleanly separate the haplotypes to produce high-quality phased assemblies. Here we describe hifiasm, a de novo assembler that takes advantage of long high-fidelity sequence reads to faithfully represent the haplotype information in a phased assembly graph. Unlike other graph-based assemblers that only aim to maintain the contiguity of one haplotype, hifiasm strives to preserve the contiguity of all haplotypes. This feature enables the development of a graph trio binning algorithm that greatly advances over standard trio binning. On three human and five nonhuman datasets, including California redwood with a ~30-Gb hexaploid genome, we show that hifiasm frequently delivers better assemblies than existing tools and consistently outperforms others on haplotype-resolved assembly.
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