Oligosaccharyltransferase (OST) catalyses the key step of N-glycosylation, transferring immature N-glycans to select Asn residues in nascent proteins in the endoplasmic reticulum (ER). Asn are more li...

Machine learning (ML) and deep learning (DL) methodologies have significantly advanced drug discovery and design in several aspects. Additionally, the integration of structure-based data has proven to...

Essential chaperones heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) collaborate in oncoprotein folding. Dual inhibition of these chap…

Post-translational modification (PTM) of proteins regulates cellular proteostasis by expanding protein functional diversity. This naturally leads to increased proteome complexity as the result of PTM ...

Antimicrobial resistance (AMR) is a global threat, with Burkholderia species contributing significantly to difficult-to-treat infections. The Pen family of β-lactamases are produced by all Burkholderi...

Screen of minimalistic enhancers in blood progenitor cells demonstrates widespread dual activator-repressor function of transcription factors (TFs) and enables the model-guided design of cell-state-sp...

Phosphodiester groups occur ubiquitously in nature, e.g. in nucleic acids or in cyclic (di-)nucleotides important for signal transduction. Proteins often use polar or positively charged amino acids t...

Nature Communications - Mutations cause impairment of function in the chaperone Hsp60. Here, the authors investigate their impact with MD simulations from the monomer to the 28-mer complex, and...

Accelerated SuFEx Click Chemistry (ASCC) is a powerful method for coupling aryl and alkyl alcohols with SuFEx-compatible functional groups. With its hallmark favorable kinetics and exceptional product...

Mutations cause impairment of function in the chaperone Hsp60. Here, the authors investigate their impact with MD simulations from the monomer to the 28-mer complex, and show the pervasive effects of ...

NADPH oxidases (NOXs) are enzymes dedicated to reactive oxygen species (ROS) production and are implicated in cancer, neuroinflammation, and neurodegenerative diseases. VAS2870 is a covalent inhibitor...

Enzymes are the quintessential green catalysts, but realizing their full potential for biotechnology typically requires improvement of their biomolecular properties. Catalysis enhancement, however, is often accompanied by impaired stability. Here, we show how the interplay between activity and stability in enzyme optimization can be efficiently addressed by coupling two recently proposed methodologies for guiding directed evolution. We first identify catalytic hotspots from chemical shift perturbations induced by transition-state-analogue binding and then use computational/phylogenetic design (FuncLib) to predict stabilizing combinations of mutations at sets of such hotspots. We test this approach on a previously designed de novo Kemp eliminase, which is already highly optimized in terms of both activity and stability. Most tested variants displayed substantially increased denaturation temperatures and purification yields. Notably, our most efficient engineered variant shows a ∼3-fold enhancement in activity (kcat ∼ 1700 s–1, kcat/KM ∼ 4.3 × 105 M–1 s–1) from an already heavily optimized starting variant, resulting in the most proficient proton-abstraction Kemp eliminase designed to date, with a catalytic efficiency on a par with naturally occurring enzymes. Molecular simulations pinpoint the origin of this catalytic enhancement as being due to the progressive elimination of a catalytically inefficient substrate conformation that is present in the original design. Remarkably, interaction network analysis identifies a significant fraction of catalytic hotspots, thus providing a computational tool which we show to be useful even for natural-enzyme engineering. Overall, our work showcases the power of dynamically guided enzyme engineering as a design principle for obtaining novel biocatalysts with tailored physicochemical properties, toward even anthropogenic reactions.

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining ...

Cell Death & Disease - Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity

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