Entropy, enthalpy, and evolution: Adaptive trade-offs in protein binding thermodynamics.

Proteins are central to biological complexity as their ligand binding processes, shaped by thermodynamics, have driven evolutionary adaptation throughout Earth's history. Despite extensive research into protein-ligand interactions, the evolution of their binding thermodynamics, particularly regarding enthalpy-entropy trade-offs, remains underexplored. This review compares experimental and computational findings to illustrate how the balance of thermodynamics influences protein structure and function over time.

Exploring Large Protein Sequence Space through Homology- and Representation-based Hierarchical Clustering.

Exploration of protein sequence space can offer insight into protein sequence-function relationships, benefitting both basic science and industrial applications. The use of sequence similarity networks is a standard method for exploring large sequence datasets, but is currently limited when scaling to very large datasets and when viewing more than one level (hierarchy) of homology. Here, we present a sequence analysis pipeline with a number of innovations that address some limitations of traditional sequence similarity networks.

Ancestral reconstruction of polyethylene terephthalate degrading cutinases reveals a rugged and unexplored sequence-fitness landscape.

The use of protein engineering to generate enzymes for the degradation of polyethylene terephthalate (PET) is a promising route for plastic recycling, yet traditional engineering approaches often fail to explore protein sequence space for optimal enzymes. In this work, we use multiplexed ancestral sequence reconstruction (mASR) to address this, exploring the evolutionary sequence space of PET-degrading cutinases. Using 20 statistically equivalent phylogenies of the bacterial cutinase family, we generated 48 ancestral sequences revealing a wide range of PETase activities, highlighting the value of mASR in uncovering functional variants.

A Thermostable Bacterial Metallohydrolase that Degrades Organophosphate Plasticizers.

A cyclase-phosphotriesterase (C-PTE) from Ruegeria pomeroyi DSS-3 has recently been identified for its capacity to detoxify several organophosphate compounds. However, several aspects of this enzyme remain unexplored, such as its activity with industrial organophosphates, its molecular structure, and its thermostability. In this work, the crystal structure of C-PTE is reported, which is solved to 2.

A F-dependent Single Domain Chemogenetic Tool for Protein De-dimerization.

Protein-protein interactions (PPIs) mediate many fundamental cellular processes. Control of PPIs through optically or chemically responsive protein domains has had a profound impact on basic research and some clinical applications. Most chemogenetic methods induce the association, i.

Computational and Experimental Exploration of Protein Fitness Landscapes: Navigating Smooth and Rugged Terrains.

Proteins evolve through complex sequence spaces, with fitness landscapes serving as a conceptual framework that links sequence to function. Fitness landscapes can be smooth, where multiple similarly accessible evolutionary paths are available, or rugged, where the presence of multiple local fitness optima complicate evolution and prediction. Indeed, many proteins, especially those with complex functions or under multiple selection pressures, exist on rugged fitness landscapes.

Synthesis, screening and validation of cysteine-reactive fragments as chikungunya virus protease inhibitors.

Alphaviruses like the Chikungunya virus cause severe outbreaks; however, no specific treatments are available. Their viral replication depends on the nsP2 cysteine protease, a promising but underexplored target for drug discovery. In this study, we report a covalent fragment screening against Chikungunya virus nsP2 protease, resulting in the identification of three inhibitors that can serve as starting points for future drug development.

Having your cake and eating it too - a new target for sporozoite-neutralising mAbs.

Current malaria vaccines provide suboptimal, waning protection. Dacon et al. identify a protective post-translationally modified epitope on Plasmodium sporozoites that is absent from current vaccines.

Isolation, identification, and characterisation of the malachite green detoxifying bacterial strain Bacillus pacificus ROC1 and the azoreductase AzrC.

Malachite green (MG) is used as a dye for materials such as wood, cotton, and nylon, and is used in aquaculture to prevent fungal and protozoan diseases. However, it is highly toxic, with carcinogenic, mutagenic, and teratogenic properties, resulting in bans worldwide. Despite this, MG is still frequently used in many countries due to its efficacy and economy.

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4-Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites.

Cyanotryptophans (CN-Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl-tRNA synthetases specific for 4-, 5-, 6-, and 7-CN-Trp for high-yield in vivo production of proteins with a single, site-specifically introduced nitrile label.

1.3 Å Crystal Structure of Peptidyl-Prolyl Isomerase B with Uniform Substitution of Valine by (2,3)-4-Fluorovaline Reveals Structure Conservation and Multiple Staggered Rotamers of CHF Groups.

(2,3)-4-Fluorovaline (FVal) is an analogue of valine, where a single CH group is substituted by a CHF group. In the absence of valine, valyl-tRNA synthetase uses FVal as a substitute, enabling the production of proteins uniformly labeled with FVal. Here, we describe the production and analysis of peptidyl-prolyl isomerase B where all 16 valine residues have been replaced by FVal synthesized with a C-labeled CHF group.

The ultra-high affinity transport proteins of ubiquitous marine bacteria.

SAR11 bacteria are the most abundant microorganisms in the surface ocean and have global biogeochemical importance. To thrive in their competitive oligotrophic environment, these bacteria rely heavily on solute-binding proteins that facilitate uptake of specific substrates via membrane transporters. The functions and properties of these transport proteins are key factors in the assimilation of dissolved organic matter and biogeochemical cycling of nutrients in the ocean, but they have remained largely inaccessible to experimental investigation.

An increased copy number of glycine decarboxylase (GLDC) associated with psychosis reduces extracellular glycine and impairs NMDA receptor function.

Glycine is an obligatory co-agonist at excitatory NMDA receptors in the brain, especially in the dentate gyrus, which has been postulated to be crucial for the development of psychotic associations and memories with psychotic content. Drugs modulating glycine levels are in clinical development for improving cognition in schizophrenia. However, the functional relevance of the regulation of glycine metabolism by endogenous enzymes is unclear.

Partitioning of an Enzyme-Polymer Surfactant Nanocomplex into Lipid-Rich Cellular Compartments Drives In Situ Hydrolysis of Organophosphates.

Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants.

Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS-CoV-2 Main Protease with Antiviral Activity.

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (M) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 M to enhance its cell permeability and in vitro antiviral activity.

Conformational Preferences of the Non-Canonical Amino Acids (2,4)-5-Fluoroleucine, (2,4)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CHF groups substitute specific methyl groups, can readily be probed by F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CHF groups is unclear. We present high-resolution crystal structures of peptidyl-prolyl - isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2,4)-5-fluoroleucine, (2,4)-5-fluoroleucine, or 5,5'-difluoroleucine.

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity.

Improving plastic degrading enzymes via directed evolution.

Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family.

Mononuclear binding and catalytic activity of europium(III) and gadolinium(III) at the active site of the model metalloenzyme phosphotriesterase.

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated.

An Engineered Biarylitide Cross-Linking P450 from RiPP Biosynthesis Generates Alternative Cyclic Peptides.

Cytochrome-P450-mediated cross-linking of ribosomally encoded peptides (RiPPs) is rapidly expanding and displays great potential for biocatalysis. Here, we demonstrate that active site engineering of the biarylitide cross-linking enzyme P450 enables the formation of His-X-Tyr and Tyr-X-Tyr cross-linked peptides, thus showing how such P450s can be further exploited to produce alternate cyclic tripeptides with controlled cross-linking states.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds dehydrogenation of a specific acylated dipeptide substrate.

WS9326A is a peptide antibiotic containing a highly unusual -methyl--2-3-dehydrotyrosine (NMet-Dht) residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase (NRPS). The cytochrome P450 encoded by (P450) has been shown to be essential for the formation of the alkene moiety in NMet-Dht, but the timing and mechanism of the P450-mediated ,-dehydrogenation of Dht remained unclear. Here, we show that the substrate of P450 is the NRPS-associated peptidyl carrier protein (PCP)-bound dipeptide intermediate ()-2-pent-1'-enyl-cinnamoyl-Thr--Me-Tyr.