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Comparing the evolvability of an ancestrally reconstructed and modern adenylate kinase

Directed evolution revolutionized the field of protein engineering by establishing a highly customizable framework to produce enzymes with enhanced catalytic power for a wide range of functions. However, modern enzymes subjected to directed evolution frequently plateau in improvement due to entrapment in local maxima on their fitness landscapes. Ancestral enzymes have been proposed as superior starting points for directed evolution due to their increased thermostability. Here we propose and e...

— Patterson, M., Ludewig, H., Wilson, C., Woldring, D. R., Kim, C., Using, A., Irvin, J., Theobald, D. L., Kern, D. 2025-12-14 00:00:00

Microfluidic Immunocapture Device for Direct Detection of Lyme Disease

Lyme Disease is a multisystem infectious disease caused by the Borellia burgdorferi complex, and is a growing threat to public health. Approximately 476,000 people are infected with Lyme in the United States each year. Although Lyme is readily treated with antibiotics when detected early, early detection remains difficult. Current testing remains difficult because the standard 2-tiered ELISA/Western assay indirectly detects Lyme via measurement of a host immune response, which suffers from an...

— Wellmerling, K. M., Kirby, B. J. 2025-12-14 00:00:00

Molecular basis of noncanonical complement C3 activation by histamine

For fifty years the tick-over mechanism has been considered responsible for priming the activation of the complement system's alternative pathway through the reaction of a nucleophilic water molecule with C3 yielding C3(H2O), even though the exclusivity of this role has been challenged by the existence of extrinsic proteases that can cleave circulating C3 into C3b. Here we show that the biogenic amine histamine can activate C3 by reacting with the internal thioester bond yielding a novel spec...

— Fernandez, F. J., de la Paz-Garcia, K., Querol-Garcia, J., Ramos-Guzman, C. A., Martin-Merinero, H., Mares-Mejia, I., Alfonso-Gonzalez, L., Rodriguez de Cordoba, S., Tunon, I., Vega, M. C. 2025-12-14 00:00:00

Uncovering the link between ATP synthase and the TCA cycle by crosslinking mass spectrometry

Mitochondrial oxidative phosphorylation (OXPHOS) comprises a series of multi-subunit protein complexes that operate in coordination with the tricarboxylic acid (TCA) cycle to generate ATP. Although these systems are metabolically interconnected, complex II is generally regarded as the only direct structural link between the OXPHOS and TCA cycle. Here, we combine in-solution crosslinking mass-spectrometry (XL-MS), quantitative proteomics, and blue native PAGE (BN-PAGE) to explore how ATP synth...

— Perez Paneda, L., Misic, J., Kadava, T., Larsson, N.-G., Heck, A. J. R. 2025-12-14 00:00:00

Regioisomer-controlled red-shifted DNA probes for imaging of living tissues

Fluorescence imaging of chromatin DNA with high resolution and specificity is key to understanding cellular processes and enabling molecular diagnostics. However, choosing the best DNA probe for in vivo imaging is often a challenging task, as systematic studies investigating the biocompatibility of these molecules are lacking. Red-shifted fluorescent probes are particularly advantageous for imaging thick tissues, as light scattering decreases at red and far-red wavelengths. We synthesized and...

— Kiszka, K. A., Pradhan, S., Bucevicius, J., Koenen, T., Lukinavicius, G. 2025-12-14 00:00:00

The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions

Bacterial non-homologous end joining (NHEJ) is a key DNA double-strand break (DSB) repair pathway that relies on the Ku-LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminal region has previously been highlighted for its role in both LigD recruitment to DNA DSBs and stimulation of LigD ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences enzymatic activities of LigD. Here, we combine NMR spectroscopy...

— Sowa, D. J., Huang, J., Marwaha, A., Subramaniam, A., Doubleday, C., Warner, M. M., Byun, J. A., Melacini, G., Andres, S. N. 2025-12-14 00:00:00

Chemical proteomics reveals regulation of bile salt hydrolases via oxidative post-translational modifications

The gut microbiome is the vast, diverse ecosystem of microorganisms that inhabits the human intestines and provides numerous essential functions for the host. One such key role is the metabolism of primary bile acids that are biosynthesized in the host liver into a plethora of secondary bile acids produced by gut bacteria. These metabolites serve as both antimicrobial and chemical signaling agents within the host. The critical microbial enzyme that plays a gatekeeping role in secondary bile a...

— Bracken, A. K., Malarney, K. P., Chang, P. V. 2025-12-14 00:00:00

Molecular basis of tRNA substrate recognition and modification by the atypical SPOUT methyltransferase Trm10

The evolutionarily conserved methyltransferase Trm10 modifies the N1 position of guanosine 9 (G9) in some tRNAs, but how the enzyme recognizes and modifies its substrate tRNAs remains unclear. Here, we used an S-adenosyl-L-methionine (SAM) analog to trap the Trm10-tRNAGly complex and enable determination of its structure in a post-catalytic state by cryogenic electron microscopy (cryo-EM). We observed three distinct complexes: two with a single Trm10 bound to tRNA that differ in their tRNA ac...

— Nandi, S., Strassler, S. E., Dey, D., Krishnamohan, A., Harris, G. M., Comstock, L. R., Jackman, J. E., Conn, G. L. 2025-12-14 00:00:00

Native, full-length, refolding-assisted purification of TDP-43 compatible with BSL-2 safety regulations

TAR DNA-binding protein 43 (TDP-43) is a prion-like RNA-binding protein that plays a key role in amyotrophic lateral sclerosis and frontotemporal dementia. Producing full-length TDP-43 consistently is thus relevant for its in vitro studies and yet it remains challenging, especially with the current requirement to work under biosafety level-2 (BSL-2) containment due to new safety regulations for Prion-like and amyloidogenic proteins. Here we describe a refolding-assisted purification protocol ...

— De Los Rios, P., dehury, s., Tiwari, S. 2025-12-14 00:00:00

Discovery of Non-Degradative Covalent Molecular Glues for Transcriptional Reprogramming

Transcriptional reprogramming through induced proximity has emerged as a powerful strategy for modulating the expression of oncogenic and tumor-suppressive genes. Inspired by transcriptional reprogramming approaches such as transcriptional/epigenetic chemical inducers of proximity (TCIPs) that link BCL6 inhibitors to transcriptional regulators, we sought to develop covalent ligands that rewire BCL6 proximity to selectively suppress MYC transcriptional output while derepressing BCL6 target loc...

— Duong, T. N., Pandji, E., Shao, Q., Nomura, D. K. 2025-12-14 00:00:00

Tryptophan Chemistry Driven by a Widespread Cytochrome P422 Enzyme Family

Tryptophan serves as a versatile biosynthetic precursor across living organisms. While heme-binding proteins (HBPs) mediate key reactions in tryptophan transformation, the full diversity of HBPs remains largely unexplored. Here, we developed the novel Cofactor-Integrative Structural Inspector (CISSspector) to systematically identify HBPs in the extensive extant microbial genomic sequence database, which revealed several uncharacterized HBP families. We experimentally characterized one of the ...

— MA, W., WANG, Q., YANG, Q., TENG, Z., HAN, X., SANG, M., LI, Q., WANG, R., FENG, P., ZHONG, J., ZHANG, Y., WEI, Y., JIANG, L., GUENGERICH, F. P., Zhang, W. 2025-12-13 00:00:00

RNA modulates FUS condensate assembly, dynamics, and aggregation through diverse molecular contacts

Fused in sarcoma (FUS) is an RNA-binding protein that undergoes phase separation with RNA and other cellular components, forming ribonucleoprotein (RNP) granules. While recent advances delineating the molecular forces that underlie phase separation have largely focused on protein-protein interactions, the molecular details of protein-RNA interactions within condensates remain limited. In this study, we demonstrate that RNA modulates the phase separation of the low-complexity (LC) and arginine...

— Zheng, T., Sojitra, K., Cummings, S., Chen, Q., Mohanty, P., Mittal, J., Fawzi, N. L. 2025-12-13 00:00:00

Single-Molecule Imaging Reveals DNA Shape Read-out by the INO80 Chromatin Remodeler

ATP-dependent chromatin remodeling enzymes act together to construct complex chromatin architectures around functionally important regulatory regions like promoters and origins of replication. The INO80 complex accurately positions nucleosomes bordering these regions, which is crucial for proper transcription start site selection and efficient replication. How INO80 localizes these nucleosomes and regulates its remodeling activity to produce specific positions mechanistically is unclear, alth...

— Stigler, J., Linder, G., Horky, D., Zernia, S. 2025-12-13 00:00:00

Using experimental results of protein design to guide biomolecular energy-function development

Computational models of macromolecules have many applications in biochemistry, but physical inaccuracies limit their utility. One class of models uses energy functions rooted in classical mechanics. The standard datasets used to train these models are limited in diversity, pointing to a need for new training data. Here, we sought to explore a new paradigm for training an energy function, where the Rosetta energy function was used to design de novo proteins. Experimental results on these desig...

— Haddox, H. K., Rocklin, G. J., Motta, F. C., Strickland, D., Halabiya, S. F., Cordray, C., Park, H., Klavins, E., Baker, D., DiMaio, F. 2025-12-13 00:00:00

Cellular responses to FGF1 are modulated by palmitoylation of the docking protein FRS2α

An important mechanism by which receptor tyrosine kinases (RTKs) mediate cellular responses involves the formation of signaling complexes through direct interactions with membrane-associated docking proteins, followed by phosphorylation of multiple tyrosine residues. These docking proteins recruit and activate downstream signaling molecules and enzymes following ligand stimulation. The docking protein FRS2 has been established as a major signaling hub activated by fibroblast growth factors (F...

— An, S., Suzuki, Y., Mohanty, J., Tome, F., Lax, I., Schlessinger, J. 2025-12-13 00:00:00

DNA binding and lesion recognition by the bacterial interstrand DNA crosslink glycosylase AlkX

Interstrand DNA crosslinks (ICLs) are a highly toxic form of DNA damage. ICL repair in both eukaryotes and bacteria involves unhooking of the two strands by specialized DNA glycosylases. We recently established that the human pathogen Acinetobacter baumannii contains an ICL glycosylase (AlkX) that facilitates pathogenesis and protects the bacteria from DNA damage and acid stress. However, the physical basis for glycosylase-catalyzed ICL unhooking is unknown. Here, we describe a crystal struct...

— Cai, Y., Kunkle, D. E., Edinbugh, M. D., Skaar, E. P., Eichman, B. F. 2025-12-13 00:00:00

Mechanism of Lutein to meso-Zeaxanthin Isomerization by RPE65 Catalysis

The macular pigments lutein (L), zeaxanthin (Z), and meso-zeaxanthin (MZ) protect the human retina from light and oxidative stress. While L and Z are abundant in the human diet, MZ is nearly absent. We previously demonstrated MZ is derived from L in chicken embryos precisely timed with expression of RPE65. Herein, we show that RPE65 from mouse, an animal that does not concentrate MZ in the eye, catalyzes L to MZ isomerization similarly as found for RPE65 from chicken and human, when expressed...

— Ballinger, A., Ranganathan, A., Bailey, M., Ramos, D., Lee, A., Li, B., Bernstein, P. S., Horvath, M. P. 2025-12-13 00:00:00

Core-shell microparticle encapsulation for pH-responsive and targeted delivery of lactoferrin and ferrous sulfate

Microgel beads of amidated low methoxy pectin and bovine lactoferrin were formed by external gelation of a water in oil emulsion with ferrous sulfate. The stability of the lactoferrin to gastric digestion and proteolysis by pepsin was determined by gel electrophoresis. The microparticles were then dispersed in chitosan and the resulting mixture was spray dried to form a shell that is insoluble at neutral pH conditions. The iron content of the microparticles without chitosan was 34 mg g-1 and ...

— Noack, C. E., Li, P., Khongkomolsakul, W., Huang, Y., Abbaspourrad, A. 2025-12-13 00:00:00

Holographic fingerprinting reveals oligomer-driven phase separation in Bovine Serum Albumin

Biomolecular condensates formed through phase separation are fundamental to cellular organization. Although the physical principles underlying intrinsically disordered proteins are well understood, the molecular determinants of condensate formation in globular proteins remain elusive. Here, we employ Holographic Particle Characterization, a label-free, high-throughput imaging technique, to investigate the self-assembly of Bovine Serum Albumin (BSA), a model globular protein. We show that this...

— Cheong, F., Lee, S. Y., Bais, S., Khanal, S., Saurabh, S. 2025-12-13 00:00:00

Nanopore sequencing reveals operon-specific ribosome remodeling accompanying naphthyridone resistance in Staphylococcus aureus

Antimicrobial resistance (AMR) threatens global health; however, the molecular adaptations underlying resistance to emerging antibiotic classes remain poorly defined. Here, we applied long-read DNA and direct RNA nanopore sequencing and developed methods to deconvolute operon-specific epitranscriptomic changes. Together, this platform uncovered a previously unrecognized, operon-specific pathway of resistance in Staphylococcus aureus to the naphthyridone antibiotic A-692345. Genomic nanopore s...

— Copoulos, D. W., Hughes, K. T., Chevance, F. F., Burrows, C. J., Fleming, A. M., Looper, R. E. 2025-12-12 00:00:00

JNJ-55511118 Stabilizes a Desensitized-like Conformation of γ8-Containing AMPA Receptors through Long-Range Allosteric Modulation

AMPA receptors (AMPARs), glutamate-gated ion channels, are dynamically regulated by auxiliary proteins such as TARP gamma 8. JNJ-55511118 (JNJ-118) is a selective inhibitor of gamma 8-containing AMPARs. The resting-state structure shows JNJ-118 binding near the transmembrane entrance at the AMPAR-gamma 8 interface, producing only localized structural effects. However, the mechanism by which JNJ-118 inhibits agonist-bound receptors remains unclear, as no activated-state structures are availabl...

— Flores, P. C., Berka, V., Jayaraman, V. 2025-12-12 00:00:00

Cryo-EM structures of anti Z-DNA antibodies in complex with antigen reveal distinct recognition modes of a left-handed geometry

Double-stranded nucleic acids can undergo transitions from canonical B/A-forms to alternate left-handed Z-DNA/Z-RNA (Z-NAs). Z-NAs are implicated in processes such as neuroinflammation in Alzheimer's disease, Lupus Erythematosus, microbial biofilms, and type I interferon-mediated human pathologies. Since endogenous Z-NA sensors like the Z domain can induce B-to-Z transitions, monoclonal antibodies (mAbs) Z-D11 and Z22 have been regarded as conformation-specific tools to confirm Z-NA in situ, ...

— Chin, D. H. R., Luo, Y., Lau, Y. T., Dutta, N., He, Z., Yin, C., Williams, R. M., Balachandran, S., Vicens, Q., Droge, P., Luo, D. 2025-12-12 00:00:00

Complete NMR assignment for 275 of the most common dipeptides in intrinsically disordered proteins

Accurate NMR chemical shift assignments are essential for atomic-resolution characterization of proteins. Especially for intrinsically disordered proteins (IDPs) and regions (IDRs), however, the assignment remains a labor-intensive task due to spectral overlap and conformational heterogeneity. Consequently, complete side-chain assignments are rare. Here, we present a comprehensive reference dataset, comprising the complete NMR chemical shift assignments for 275 of the most prevalent dipeptide...

— Rindfleisch, T., Taule, E. F., Miettinen, M. S., Underhaug, J. 2025-12-12 00:00:00

Deacetylation of β-Mannans by Two Complementary Carbohydrate Esterases from the Human Gut Microbe Bacteroides cellulosilyticus

{beta}-Mannans are widespread in the human diet as components of plant-derived foods and as food additives. Several classes of {beta}-mannans are decorated with acetylations, which are key for their physicochemical properties and protection against enzymatic degradation. While the process for depolymerization of acetylated {beta}-mannans has been described in depth for members of the phylum Bacillota, there is limited mechanistic knowledge on how Bacteroidota utilize these glycans. Here, we c...

— Lindstad, L. J., Mrozek, P. M., Boehlich, J., Leivers, S., Pope, P., La Rosa, S. L., Westereng, B. 2025-12-12 00:00:00

A Highly Selective, Cell-Permeable Fluorescent Probe for Imaging Histone Deacetylase 6 in Live Cells

Histone Deacetylase 6 (HDAC6) plays a crucial role in diverse cellular processes, including cytoskeletal regulation, protein quality control, and stress responses; and its dysregulation is linked to multiple cancers and neurodegenerative disorders, making it a key therapeutic target. However, a detailed understanding of its dynamic functions has been limited by the lack of chemical tools for its visualization in living systems. Utilizing the highly selective inhibitor Nexturastat A as a targe...

— Nguyen, V. T., Koenen, T., Bucevicius, J., Lukinavicius, G. 2025-12-12 00:00:00

Evaluation of peptidomimetic inhibitors

Scientific research in drug discovery relies on robust, versatile, and well-established methodologies. One effective strategy for targeting disease-relevant proteins, such as E3 ligases, involves rational drug design using degron peptides as starting points for compound development. Here, we present a comprehensive set of complementary assays for the evaluation of degron-based binders, enabling their integration into an efficient drug discovery pipeline. We introduce screening strategies such...

— Lenz, C., Knapp, S., Saxena, K. 2025-12-12 00:00:00

Tissue-guided multi-omics profiling identifies extracellular vesicle biomarkers indicative of lung pathology in acute respiratory distress syndrome

Background: Acute respiratory distress syndrome (ARDS) remains a lethal inflammatory lung condition lacking reliable biomarkers that reflect lung-specific pathology. Extracellular vesicles (EVs) circulate systemically and may carry molecular signals from injured organs, but the correspondence between EV cargo and lung tissue alterations remains unclear. Methods: We established aspiration-, lipopolysaccharide (LPS)-, and COVID-19-induced murine ARDS models and applied a tissue-guided multiomic...

— Chen, P.-T., Chang, C.-Y., Chung, C.-L., Lee, C.-H., Suk, C.-W., Lin, C.-F., Fan, Y.-J., Lu, Y.-W., Hsu, Y.-C., Chang, T., Huang, C.-J., Tsai, I.-L. 2025-12-12 00:00:00

Stereoselective Degradation of Diacylglycerol Kinases Potentiate T cell Activation and Tumor Cell Cytotoxicity

Stereoselective recognition is a powerful means to differentiate selective versus non-specific activity of small molecules in complex biological systems. Here, we disclose stereochemically defined, sulfonyl-triazole inhibitors of the lipid enzyme diacylglycerol kinase-alpha (DGKA), a key metabolic checkpoint for T cell effector function. Acute treatment with the covalent DGKA inhibitor AHL-7160 recruited endogenous DGKA to the plasma membrane in a stereoselective and isozyme-specific manner. ...

— Shaikh, M., Mookherjee, S. P., Weckerly, C., Libby, A. H., Xiao, A., Zhao, Y., Vaidya, S. D., Kim, A., Li, Z., Ware, M. L., Marants, M., Murtagh, O., Wolfe, W. J., Bullock, T. N., Purow, B. W., Hammond, G. R., Hsu, K. 2025-12-12 00:00:00

Small Molecule Degradation of the microRNA-21 Precursor Rescues Pathogenic Pathways in Cellular Models of Fibrosis

MicroRNAs (miRNAs) are short RNA molecules that bind to target mRNAs, resulting in translational repression and gene silencing. Overexpression of microRNA-21 (miR-21) is associated with various human diseases, including autosomal dominant polycystic kidney disease (ADPKD) and pulmonary fibrosis. In this study, a previously described heterobifunctional molecule, TGP-21-RiboTAC, that degrades the miR-21 precursor (pre-miR-21) in triple negative breast cancer cells was investigated in polycystic...

— Wang, T., Yang, X., Li, Y., Song, J. H., Disney, J. L., Garcia, J. G. N., Disney, M. D. 2025-12-12 00:00:00

Engineering SIRPα conformational plasticity to reveal a cryptic pocket suitable for structure-based drug design

The protein-protein interaction between Signal Regulatory Protein alpha (SIRP) and CD47 is a critical immune checkpoint that enables tumor immune escape, making it a key target for cancer immunotherapy. While antibody-based therapies exist, the development of small-molecule inhibitors has been hindered by the flat, featureless binding interface. Here, we report the discovery of a novel, druggable cryptic pocket within the SIRP D1 domain (the WYF pocket), revealed through a structure-based fra...

— Storder, M., Barelier, S., Cordier, F., YACOUB, T., Ilari, L., Barral, K., MAHMOODI, S., Saez-Ayala, M., Combes, S., Betzi, S., Ulliana, A., Torres, F., Rubin, J., Roche, P., Morelli, X., Garcin, E. D., Miller, T. W. 2025-12-11 00:00:00