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<title>bioRxiv Subject Collection: Biochemistry</title>
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This feed contains articles for bioRxiv Subject Collection "Biochemistry"
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<link>https://www.biorxiv.org</link>
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<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.29.735100v1?rss=1">
<title>
<![CDATA[
Structural basis of the nucleotide incorporation cycle of bacterial DNA polymerase III 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.29.735100v1?rss=1
</link>
<description><![CDATA[
Bacterial DNA polymerase III (PolIII) is the primary replicative polymerase responsible for faithful genome duplication, yet high-resolution structural snapshots of the enzyme during DNA synthesis and proofreading have remained elusive. Here we determine cryo-EM structures of the Escherichia coli PolIII core-clamp-DNA binary, ternary and proofreading complexes at 2.4-2.7 [A] resolution, revealing the structural basis of nucleotide selection, incorporation and proofreading. Binding of an incoming nucleotide expels the single-stranded template overhang from the polymerase central channel and induces a large rotation of the polymerase index finger, which closes around the incoming nucleotide to form a compact nascent base-pair-binding pocket. A conserved tyrosine on the finger stacks against the incoming nucleotide to impose stringent steric constraints that promote nucleotide selection, whereas a conserved histidine acts as a steric gate to exclude ribonucleotides. The proofreading structures capture key intermediates that reveal how PolIII transfers a mismatched primer terminus to the 3'-5' exonuclease active site and excises the misincorporated nucleotide while avoiding unnecessary degradation of correctly paired DNA. Together, these findings define the molecular mechanism by which the bacterial replicative polymerase couples rapid and processive DNA synthesis with exceptional replication fidelity.
]]></description>
<dc:creator><![CDATA[ Xu, Z.-Q., Jergic, S., Zhu, Q., Yu, H., Lewis, J. S., Brown, S. H., Bouwer, J., Ghodke, H., Lewis, P. J., Tolun, G., Oakley, A., Dixon, N. E. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.29.735100</dc:identifier>
<dc:title><![CDATA[Structural basis of the nucleotide incorporation cycle of bacterial DNA polymerase III]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734872v1?rss=1">
<title>
<![CDATA[
Sequential Penta-Omic Extraction Method Using Single Biospecimens of Post-mortem Human Brain 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734872v1?rss=1
</link>
<description><![CDATA[
A multi-omic approach utilizing a single biospecimen is important to avoid intra-sample heterogeneity associated with testing multiple omic single-samples, and for more efficient use of small volumes of precious biopsies (<30 mg). This is especially true for the microanatomy of post-mortem human brain samples. Using post-mortem human brain biospecimens from the NIH NeuroBioBank, a penta-omic sequential extraction method is described, Simultaneous Metabolomic, Proteomic, Lipidomic, DNA, RNA Extraction (SiMPL-DREx). Each sequential omic extract was compared to those obtained by the gold standard single omic method. Preserving RIN is critical for brain and tissue banks, as it is a primary measure of tissue quality. For all five omic extracts, the tissue integrity numbers and omic profiles did not significantly differ from those obtained by the respective omic gold standard method. Unlike past multi-omic studies, this study quantified the relative solvent percentages and upstream losses for both the organic and aqueous phases, confirming an omics loss of under 5%.
]]></description>
<dc:creator><![CDATA[ Lyon, S. P., Ehrmann, B. M., Webb, T. S., Arciniega, C., Herring, L. E., Guo, S., Parnham, S., Scott, W. K., Mieczkowski, P. A., Macdonald, J. M. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734872</dc:identifier>
<dc:title><![CDATA[Sequential Penta-Omic Extraction Method Using Single Biospecimens of Post-mortem Human Brain]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734651v1?rss=1">
<title>
<![CDATA[
Intact and single-molecule analysis of heparan sulfate 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734651v1?rss=1
</link>
<description><![CDATA[
Establishing tools to couple biological processes to a DNA sequence has transformed our ability to monitor life at the molecular scale due to the scalability, flexibility, and low cost of DNA sequencing. Key examples include DNA-protein (ChIP-seq), RNA-protein (CLIP-seq), protein-protein (proximity ligation assay), and Cas-based recording of cellular events. In contrast, this paradigm has not yet significantly enhanced studies of glycans, which are mostly limited to non-DNA based chemical and biochemical assays. While classical asparagine-linked and serine/threonine-linked glycans can be directly sequenced using mass spectrometry, glycosaminoglycans - notable players in the extracellular matrix - cannot be easily analyzed in their full-length form. Here we introduce HS-nano-seq, a generalized framework to selectively label, process, and detect features of heparan sulfate on a nanopore sequencing platform. Recognizing that heparan sulfate is biochemically analogous to a nucleic acid, we report purification techniques using rapid nucleic acid strategies and conjugation methods to couple DNA adapters, generating HS-DNA chimeras resolved as discrete species by capillary electrophoresis (CE). The CE assay can distinguish features of chain length and sulfation patterns. At the single-molecule level enabled by nanopore sensing, we classify a library of synthetic heparan sulfate standards and demonstrate that nanopore ionic current fingerprints encode sulfation-dependent structural features of individual HS chains. Analysis of intact, cell-derived HS could discriminate features of individual chains with different sulfation patterns, defining the heterogeneity of binding motifs across cell types and how cells organize and program the tethered extracellular matrix. More broadly, HS-nano-seq establishes a framework for achieving full-length readouts of ECM glycopolymers that are amenable to the same biological interrogation as nucleic acids.
]]></description>
<dc:creator><![CDATA[ Hristov, P., Kakhaki, P. D., Tzadikario, T., Rai, S. K., Su, G., Olivieri, P. H., Esko, J. D., Liu, J., Jain, M., Flynn, R. A. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734651</dc:identifier>
<dc:title><![CDATA[Intact and single-molecule analysis of heparan sulfate]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734858v1?rss=1">
<title>
<![CDATA[
CAGE-TRX expands the scope of time-resolved crystallography through genetically encoded active-site photocaging 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734858v1?rss=1
</link>
<description><![CDATA[
We present CAGE-TRX, a broadly applicable time-resolved strategy for pump/release-quench-probe cryocrystallography and pump/release-probe room temperature serial crystallography. These workflows enable light-triggered control of enzyme activity via genetically encoded photocaged amino acids. By decoupling reaction initiation from substrate design, this approach allows synchronized catalysis in crystallo and the capture of transient intermediates. Using {beta}-lactamases as model systems, we demonstrate efficient decaging, restoration of activity, and structural visualization of reaction intermediates.
]]></description>
<dc:creator><![CDATA[ Smith, C., Maggiolo, A. O., Jonosko, C., Charette, M. E., Paul, N., Toth, M., Calero, G., Carr, S. M., Russi, S., Vakulenko, S. B., Deiters, A., Cohen, A. E. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734858</dc:identifier>
<dc:title><![CDATA[CAGE-TRX expands the scope of time-resolved crystallography through genetically encoded active-site photocaging]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734905v1?rss=1">
<title>
<![CDATA[
In Vivo Quantification of Histone Acetylation Turnover and Acetyl-CoA Sources Using 2H2O Metabolic Labeling and High-Resolution Mass Spectrometry. 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734905v1?rss=1
</link>
<description><![CDATA[
Dysregulated histone acetylation links cellular metabolism to gene expression, but measuring its in vivo turnover remains technically challenging. Here, we introduce a 2H2O-based metabolic labeling method coupled with high-resolution Orbitrap mass spectrometry to quantify in vivo histone acetylation dynamics. The approach leverages differing deuterium incorporation rates between fast-labeling acetyl groups and slow-labeling peptide backbones. A two-tier analytical workflow uses full-scan mass spectrometry for mono-acetylated peptides, combined with parallel reaction monitoring (PRM) to resolve site-specific turnover and stoichiometry. Furthermore, monitoring acetyl-group plateau 2H enrichment enables the evaluation of specific substrate contributions to the acetyl-CoA pool supporting histone acetylation. To demonstrate biological utility, we applied this approach to mice maintained on a high-carbohydrate diet or subjected to 48-h fasting to assess nutrient-dependent histone acetylation dynamics. Acetyl-group labeling reflected the metabolic origin of acetyl-CoA, showing greater 2H enrichment in the fed state and reduced enrichment during fasting due to increased utilization of unlabeled fatty acid-derived acetyl-CoA. Fasting accelerated acetylation turnover across multiple histone sites and reduced overall acetylation stoichiometry. Quantitative tracing revealed that fatty acid oxidation becomes an important contributor to histone acetylation during fasting, whereas glucose remains the predominant source of nucleo-cytosolic acetyl-CoA (supplying > 60% of acetylation used carbon). This approach enables simultaneous in vivo assessment of histone acetylation turnover, site occupancy, and acetyl-CoA substrate utilization, offering a robust platform to investigate metabolic-epigenetic crosstalk in health and disease.
]]></description>
<dc:creator><![CDATA[ Arias-Alvarado, A., Sabir, U., Ilchenko, S., Parrish, S., Aghayev, M., He, W., Tsai, T.-H., Zhang, G., Kasumov, T. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734905</dc:identifier>
<dc:title><![CDATA[In Vivo Quantification of Histone Acetylation Turnover and Acetyl-CoA Sources Using 2H2O Metabolic Labeling and High-Resolution Mass Spectrometry.]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734850v1?rss=1">
<title>
<![CDATA[
Incorporating Surfaced-Induced Dissociation Mass Spectrometry Data into an AlphaFold-derived deep learning network improves protein structure prediction 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734850v1?rss=1
</link>
<description><![CDATA[
Surface-Induced Dissociation native Mass Spectrometry (SID-nMS) is a tandem MS activation method that yields information on the connectivity and stoichiometry of protein complexes. While insufficient for direct structure elucidation, the data derived from SID-nMS has considerable potential to inform multimeric protein structure prediction. We hypothesized that incorporating this data into a machine-learning framework could improve multimer prediction accuracy beyond that of existing deep-learning methods. To this end, we developed SIDFold, a novel AlphaFold-based deep-learning network. SIDFold is the first AlphaFold-like network to leverage experimental data during protein complex prediction, and the first deep-learning network to utilize nMS data for structure prediction. We benchmarked SIDFold on the BETA protein set, and observed an improvement in RMSD in 138 of 227 cases including 27 targets in which the predicted structure attained near-native accuracy. We then evaluated the network on 20 proteins with experimental SID-nMS data, yielding an improved RMSD in 18 cases, with five of these cases improving to a high-accuracy complex. Finally, we tested SIDFold against a previously published SID-guided Rosetta docking method, where we saw improvement in 13 of 16 proteins. SIDFold is freely available on GitHub, with example files and commands available in the Supplementary Information.
]]></description>
<dc:creator><![CDATA[ Bolz, R. M., Day, E. H., Drake, Z. C., Harvey, S. R., Wysocki, V. H., Lindert, S. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734850</dc:identifier>
<dc:title><![CDATA[Incorporating Surfaced-Induced Dissociation Mass Spectrometry Data into an AlphaFold-derived deep learning network improves protein structure prediction]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734871v1?rss=1">
<title>
<![CDATA[
Molecular mechanism of IF1- and IF2-driven translation initiation in bacteria 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734871v1?rss=1
</link>
<description><![CDATA[
Bacterial translation initiation is a highly regulated process essential for accurate start codon selection and the assembly of an elongation-competent ribosome. Two initiation factors, 1 (IF1) and 3 (IF3), contribute to quality control for formation of 30S preinitiation complex (30S PIC), while the GTPase IF2 facilitates stable initiator tRNA binding and promotes subunit association. However, the molecular mechanism of IF1 action and the regulatory role of IF2-mediated GTP hydrolysis and inorganic phosphate (Pi) release remain poorly understood. Using ensemble cryo-EM integrated with fast-kinetics, we delineate the translation initiation pathway involving IF1 and IF2. We show that IF1 transiently associates with the 30S subunit and interferes with the formation of multiple inter-subunit bridges. IF2 promotes subunit association by stabilizing the 30S PIC through interactions mediated by its N-terminal domains. IF1 departure happens after or concomitant with GTP hydrolysis, following which the inter-subunit bridges establish. Then Pi release triggers remodeling of IF2 followed by its departure from the 70S initiation complex. These findings reveal how the coordinated interplay of IF1 and IF2 with the ribosome ensures translational fidelity and plays crucial role for formation of elongation-competent 70S.
]]></description>
<dc:creator><![CDATA[ Guerra, G. S., Zafar, H., Ge, X., Basu, R., Huang, C., Hassan, A., Valdez, N., Brabencova, S., Slamova, L., Mandava, C. S., Gamper, H., Hou, Y.-M., Gagnon, M., Sanyal, S., Demo, G. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734871</dc:identifier>
<dc:title><![CDATA[Molecular mechanism of IF1- and IF2-driven translation initiation in bacteria]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734839v1?rss=1">
<title>
<![CDATA[
Selective knockout of PKA regulatory subunits reveal opposite catalytic and metabolic consequences with implications for Alzheimer's disease 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734839v1?rss=1
</link>
<description><![CDATA[
cAMP-dependent Protein Kinase A (PKA) is a master regulator of cell signaling involved in energy metabolism, synaptic plasticity, and stress response. Dysregulated PKA signaling is implicated in diseases including neurodegeneration and cancer. The catalytic activity of PKA is regulated by two nonredundant regulatory subunits, Type I (RI/RI{beta}) and Type II (RII/RII{beta}), whose divergent functions are not fully understood. We generated double-knockout (KO) cell lines of RI/RI{beta} and RII/RII{beta} subunits and performed multiplexed MS-based proteomic and phosphoproteomic profiling under basal and glucose-perturbed conditions. We found that RI and RII loss drives distinct, and often opposite, remodeling of the cellular proteome and phosphoproteome. While both mutants blunted metabolic flexibility to glycolytic stressors and stimuli, RI and RII KO cells exhibited elevated and depressed glycolytic signaling, respectively. Interestingly, RI KO increased the abundance kinase activity of the PKA catalytic subunit C isoform, leading to an increase in PKA substrate phosphorylation, whereas RII KO decreased the abundance, kinase activity, and substrate phosphorylation by the catalytic subunit C{beta} isoform. Notably, one of the most differentially affected PKA sites between RI and RII KOs maps to the microtubule-associated protein Tau (MAPT), whose hyperphosphorylation is a hallmark of Alzheimer's disease. Loss of RI increased Tau phosphorylation, which was not only caused by increased PKA catalytic activity, but also a higher binding affinity of Tau to RII subunits on the negatively-charged flexible linker region. Overall, the present study demonstrates that PKA RI and RII subunits play nonredundant roles in modulating PKA activity, metabolic flexibility, and phospho-regulation of key disease-associated substrates such as Tau.
]]></description>
<dc:creator><![CDATA[ Rossitto, L.-A. M., Lu, T., Ma, Y., Kaila Sharma, P., Burghi, V., Gonzalez, C. C., Bruystens, J., Maurya, S., Wu, J., Lona, A., Kufareva, I., Gutkind, J. S., Gonzalez, D. J., Chen, X., Taylor, S. S. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734839</dc:identifier>
<dc:title><![CDATA[Selective knockout of PKA regulatory subunits reveal opposite catalytic and metabolic consequences with implications for Alzheimer's disease]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734876v1?rss=1">
<title>
<![CDATA[
Genetic Code Expansion for Site-Specific Encoding of a Switchable, Intrinsic Fluorophore-Quencher Pair to Monitor Protein Dynamics 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734876v1?rss=1
</link>
<description><![CDATA[
Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore-quencher pair comprising two non-canonical amino acids - acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet) - into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by biorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern-Volmer quenching, fluorescence lifetime measurements, and "proline ruler" peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: 1) calmodulin, a calcium-sensing protein, 2) RecA, a DNA damage sensor in bacteria, and 3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, enables high-throughput drug screening, and advances tools for studying protein structure-function relationships.
]]></description>
<dc:creator><![CDATA[ Giri, P., Yarra, V., Mathis, M., Hurley, C., Jones, C., Eteme, O. N., Hostetler, Z., Cooley, R. B., Kohli, R., Mehl, R., Petersson, E. J. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734876</dc:identifier>
<dc:title><![CDATA[Genetic Code Expansion for Site-Specific Encoding of a Switchable, Intrinsic Fluorophore-Quencher Pair to Monitor Protein Dynamics]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734730v1?rss=1">
<title>
<![CDATA[
Lipid-Coated Water-in-Oil Droplets as a Passivation-Free Platform for Cost-Effective Fluorescence Spectroscopy 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734730v1?rss=1
</link>
<description><![CDATA[
Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications.
]]></description>
<dc:creator><![CDATA[ Trowbridge, J. W., Lakic, A., Brodbeck, A., Cox, D., Mason, A. F., McAlary, L. ]]></dc:creator>
<dc:date>2026-06-29</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734730</dc:identifier>
<dc:title><![CDATA[Lipid-Coated Water-in-Oil Droplets as a Passivation-Free Platform for Cost-Effective Fluorescence Spectroscopy]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-29</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734512v1?rss=1">
<title>
<![CDATA[
Where do the ligands bind? Co-folding bitter taste GPCRs with the BitterDB chemical space 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734512v1?rss=1
</link>
<description><![CDATA[
Bitterness is a key taste modality mediated in vertebrates by TAS2R G protein coupled receptors, which also function in diverse extraoral tissues. Recent cryo EM structures have revealed a non classical intracellular pocket in TAS2R14, raising the question of whether ligand pocket choice can be predicted computationally and what sequence features control it. Here we evaluate the Boltz 2 co folding framework on all currently available agonist-TAS2R cryo EM complexes and show that it correctly identifies the experimentally observed binding pocket for 12 of 15 pairs, including intracellular binding that docking into predicted receptor models fails to reproduce. Focusing on aristolochic acid, which binds intracellularly to TAS2R14 and extracellularly to TAS2R43, we use a series of in silico morphing experiments to pinpoint transmembrane helices 3 and 7, and specific residues within them, as key determinants of pocket preference. Extending the analysis to ~1,500 agonist-receptor associations from BitterDB, we find that while most receptors are predicted to bind agonists predominantly in the extracellular pocket, several TAS2Rs may have both extracellularly and intracellularly binding ligands. Finally, by fine tuning the Boltz 2 affinity module on ~7,000 positive and negative experimental data points, we obtain a TAS2R specific classifier that improves AUROC from 0.54 to 0.82 and average precision from 0.24 to 0.58 on a validation set.
]]></description>
<dc:creator><![CDATA[ Ziaikin, E., Niv, M. Y. ]]></dc:creator>
<dc:date>2026-06-27</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734512</dc:identifier>
<dc:title><![CDATA[Where do the ligands bind? Co-folding bitter taste GPCRs with the BitterDB chemical space]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-27</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734764v1?rss=1">
<title>
<![CDATA[
Cryo-electron microscopy structure of the bovine ephemeral fever virus RNA-nucleoprotein assembly 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734764v1?rss=1
</link>
<description><![CDATA[
Bovine ephemeral fever virus (BEFV), a member of the Rhabdoviridae family, is an arthropod-borne pathogen that causes acute febrile disease in cattle. The structural basis of its genome encapsidation and virion assembly remains unexplored, with the current knowledge largely limited to predictions derived from bioinformatic comparisons with other rhabdoviruses. Furthermore, the structural principles that permit the formation of variable-diameter nucleocapsids, resulting in the distinctive bullet-shaped morphology of rhabdoviruses, remain poorly understood. Here, we report the cryoelectron microscopy structure of the BEFV nucleoprotein (N) in complex with RNA, in the absence of other viral components. The complex predominantly forms circular decameric oligomers that we propose to act as nucleation intermediates during assembly of the bullet-shaped nucleocapsids. Direct subunit interactions are limited to a small polar surface area, with additional intersubunit links mediated by flexible N- and C-terminal loops. These interfaces generate a structurally plastic oligomeric lattice in which neighbouring N subunits can undergo substantial rigid-body rotations and positional rearrangements while preserving conserved local contacts and continuous RNA encapsidation. Such quasi-equivalent interactions provide a plausible mechanism for accommodating the progressive changes in helical diameter required for the transition from the highly curved bullet tip to the wider cylindrical trunk of rhabdovirus nucleocapsids. The assembly is stabilised by the bound RNA molecule, where nine RNA bases are accommodated by each N subunit. The RNA-binding mechanism is consistent with that of VSV, the closest BEFV homologue characterised structurally, but differs at about half of the RNA-binding residues, demonstrating the versatility of the nucleoprotein scaffold in interacting with ssRNA. Comparative analysis with other rhabdoviruses, as well as negative-sense RNA viruses with constant-diameter nucleocapsids, such as Ebola, further confirms the structural features that enable bullet-shaped versus cylindrical nucleocapsid assembly.
]]></description>
<dc:creator><![CDATA[ Herman, A., Antson, A. A., Bardy, P. ]]></dc:creator>
<dc:date>2026-06-27</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734764</dc:identifier>
<dc:title><![CDATA[Cryo-electron microscopy structure of the bovine ephemeral fever virus RNA-nucleoprotein assembly]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-27</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734570v1?rss=1">
<title>
<![CDATA[
Systematic optimization and benchmarking of synchro-PASEF for high-throughput phosphoproteome profiling 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734570v1?rss=1
</link>
<description><![CDATA[
High-throughput data-independent acquisition (DIA) workflows paired with short chromatographic separations are increasingly adopted for systems biology and clinical proteomics. However, narrower peak widths from rapid separations demand faster mass spectrometer cycle times to maintain quantitative depth and reproducibility. The synchro-PASEF acquisition mode on timsTOF mass spectrometers diagonally scans across ion mobility and m/z space, enabling efficient sampling of the precursor ion cloud with shortened cycle times. While synchro-PASEF has demonstrated competitive identification depth for global protein abundance samples compared to conventional dia-PASEF, its performance for phosphoproteomics - where the precursor ion cloud is characteristically broader and bimodally distributed - has not been evaluated. Here, we systematically optimized synchro-PASEF methods for phosphoproteomics and benchmarked performance against two dia-PASEF methods across three sub-hour separations. We found that synchro-PASEF performance depends critically on balancing diagonal window number, total isolation width, and gradient length, with longer gradients favoring more windows for selectivity and shorter gradients favoring fewer windows to preserve sampling frequency. An optimized configuration quantified over 19,000 localized phosphosites using a 23-minute separation. Retention time summation (RTsum) with a factor of 2 increased phosphopeptide identifications by 5-20% and reduced phosphosite-level coefficients of variation by up to 30% across all dia-PASEF and synchro-PASEF methods tested. Using {beta}2-adrenergic receptor (B2AR) activation as a signaling model, we demonstrate that label-free DIA phosphoproteomics can be used to model phosphoproteomics dose-response relationships, showing that synchro-PASEF and dia-PASEF produce highly concordant phosphoproteomic responses, with comparable numbers of responding phosphosites, similar effect sizes, and nearly identical predicted protein kinase A (PKA) substrates downstream of the activated B2AR. While synchro-PASEF did not surpass optimized dia-PASEF in identification depth, its comparable biological performance and amenability to post-acquisition optimization through RTsum support its utility for high-throughput phosphoproteomics. This work provides a transferable framework for synchro-PASEF method optimization and demonstrates the broad utility of retention time summation for PASEF-based phosphoproteomics workflows.
]]></description>
<dc:creator><![CDATA[ Brademan, D., Mullarkey, A., Greeson, M., Szvetecz, S., Vitek, O., Blythe, E., Huttenhain, R. ]]></dc:creator>
<dc:date>2026-06-27</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734570</dc:identifier>
<dc:title><![CDATA[Systematic optimization and benchmarking of synchro-PASEF for high-throughput phosphoproteome profiling]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-27</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734742v1?rss=1">
<title>
<![CDATA[
Depleting prion protein using splice-switching small molecules 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734742v1?rss=1
</link>
<description><![CDATA[
Prion diseases are fatal neurodegenerative disorders driven by prion protein (PrP) misfolding, and lowering cellular PrP represents a promising therapeutic strategy. Here we report a small-molecule approach that reduces PrP by modulating pre-mRNA splicing of the PRNP gene. Through chemical modification of the clinically approved splicing modulator risdiplam, we generated CP3, the first class of compounds that selectively activate a cryptic exon in PRNP and routes its mRNA product for degradation, reducing PrP by ~70% in cells. We demonstrate that CP3 activity critically depends on alternative splicing factor Luc7L, revealing a novel requirement for alternative splicing factors in small molecule splicing modulation. Strikingly, co-administration of CP3 with a Luc7L activator PTC258 significantly lowers PrP levels in the brains of transgenic mice. These results establish splicing modulation as a powerful strategy for PrP reduction and highlight the potential of small-molecule cooperativity for therapeutic RNA targeting.
]]></description>
<dc:creator><![CDATA[ Liu, B., Fair, B. J., Kuang, Z., Tang, Z., Zhao, J., Zhou, L., Kong, Q., Solanki, A., Kenny, C., Mastrianni, J. A., Zhao, R., Li, Y., Wang, J. ]]></dc:creator>
<dc:date>2026-06-27</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734742</dc:identifier>
<dc:title><![CDATA[Depleting prion protein using splice-switching small molecules]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-27</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.734721v1?rss=1">
<title>
<![CDATA[
Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.734721v1?rss=1
</link>
<description><![CDATA[
Cellular respiration depends on transferring electrons to hydrophobic quinones in membrane bilayers, constraining capacity to available surface area. Expanding this capacity is thought to have driven cellular complexity and eukaryogenesis, with Gram-negative bacteria evolving internal invaginations and eukaryotes using membrane-bound organelles. Whether Gram-positive bacteria, which lack such membranes, evolved alternatives was unknown. Here, we show that Bacillus subtilis forms a quinone-transporting pseudomembrane composed of filaments of the NADH dehydrogenase Ndh and the quinone-transporting protein Ncp. Cryo-EM, lipidomics, and molecular dynamics reveal that Ndh and Ncp co-assemble with phospholipids into a complex containing a solvent-excluded hydrophobic lumen that sequesters quinones. These complexes further assemble into filaments, linking chambers into a continuous conduit that amplifies quinone reduction while occupying minimal membrane space. Phylogenetic analysis suggests this recent innovation is widespread in Bacillota. Quinone-transporting filaments thus reveal a third strategy for overcoming surface-area limits and provide principles for engineering synthetic energy systems.
]]></description>
<dc:creator><![CDATA[ Kropp, A., Asadollahi, K., Stapleton, J. A., Simsive, L., Leung, P. M., Darnell, R. L., Barlow, C., Hartmann, B. G., Yates, N. D. J., Fox, D. R., Greening, C., Zdorevskyi, O., Sharma, V., Blaza, J. N., Parkin, A., Grinter, R. ]]></dc:creator>
<dc:date>2026-06-27</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.734721</dc:identifier>
<dc:title><![CDATA[Quinone-transporting filaments extend the respiratory chain of Gram positive bacteria]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-27</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734504v1?rss=1">
<title>
<![CDATA[
Electron confurcation drives photosynthetic H2 production in cyanobacteria 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734504v1?rss=1
</link>
<description><![CDATA[
Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H2 uptake, HoxEFUYH utilizes NAD+ and oxidized ferredoxin, whereas H2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from dark carbohydrate oxidation. This requires reassessment of current strategies for green H2 production in cyanobacteria.
]]></description>
<dc:creator><![CDATA[ Mahapatra, G. P., Strabel, N., Lorent, C., Kumar, A., Bohn, S., Klamke, M. A., Boehm, M., Teutloff, C., Zebger, I., Appel, J., Schuller, J., Gutekunst, K. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734504</dc:identifier>
<dc:title><![CDATA[Electron confurcation drives photosynthetic H2 production in cyanobacteria]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734600v1?rss=1">
<title>
<![CDATA[
Cardiolipin modulation of MmpL3 in mycobacteria 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734600v1?rss=1
</link>
<description><![CDATA[
The complex cell envelope of mycobacteria is characterized by the presence of a unique outer membrane (OM) rich in mycolic acids (MAs). These long-chain, branched fatty acids confer extreme hydrophobicity to the OM, in part rendering the mycobacterial envelope impermeable to antibiotics and host defences. How MAs are transported from the inner membrane (IM) to the OM is largely unknown. The integral membrane protein MmpL3 plays an essential role in this process, but the mechanism(s) by which it exploits the proton motive force to flip and/or release MAs at the IM, in the form of trehalose monomycolates (TMMs), remains elusive. Here, we reconstitute and quantify the proton translocation activity of MmpL3 in artificial lipid bilayers, and discover a novel role for the phospholipid species, cardiolipin (CL), in regulating MmpL3 function. We find that mutations in conserved residues, or binding of known inhibitors in the central channel of MmpL3 do not diminish proton translocation activity. Instead, the specific presence of CL abolishes proton translocation by MmpL3 in vitro. Furthermore, we establish that an MmpL3 variant containing substitutions in a CL-binding site predicted in silico is no longer modulated by CL in vitro, and is unable to support growth of Mycobacterium smegmatis. Our work provides previously unappreciated insights into lipid regulation of MmpL3 activity in mycobacteria, and expands the guiding principles for the development of anti-mycobacterial inhibitors targeting this essential transporter.

SignificanceMycobacterial species, including the human pathogen Mycobacterium tuberculosis, are surrounded by a double-membrane cell envelope that makes them intrinsically resistant to many antibiotics. Specifically, the outer membrane (OM) contains unique lipids called mycolic acids (MAs), whose transport pathway across the envelope is poorly understood. In this study, we characterized the biochemical activity of the essential MA transporter MmpL3, and uncovered a novel mechanism for lipid-mediated functional regulation. Our work highlights the importance of protein-lipid interactions in defining transporter activity, provides insights into MA transport and OM assembly in mycobacteria, and sets the stage for the development of anti-mycobacterial strategies.
]]></description>
<dc:creator><![CDATA[ Li, S., Brown, C. M., Sun, R. H., Stansfeld, P. J., Chng, S.-S. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734600</dc:identifier>
<dc:title><![CDATA[Cardiolipin modulation of MmpL3 in mycobacteria]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734654v1?rss=1">
<title>
<![CDATA[
Evolution of a core ribosomal innovation in octopus 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734654v1?rss=1
</link>
<description><![CDATA[
Much of biology focuses on how genetic changes mediate new functions, but less attention is given to adaptations in other steps of the central dogma. Octopuses exhibit complex nervous systems and sophisticated behaviors that rival vertebrates, but via an entirely divergent evolutionary history. Here, we serendipitously discovered that octopus ribosomes contain a structural break in the core ribosomal RNA that is unique among all animals. This break site enhances translation fidelity to reduce miscoding and subsequent protein aggregation, even when engineered into evolutionarily distant bacterial ribosomes. Furthermore, high fidelity translation by octopus ribosomes supports proteomic stability during extensive RNA editing observed in cephalopods, suggesting synergy between distinct non-canonical modes of gene regulation. This adaptation emerged in recently derived octopuses with expanded nervous systems, thereby revealing a mechanism that could broadly support the evolution of novel organismal traits.
]]></description>
<dc:creator><![CDATA[ Mitra, R., Han, R., Scott, T. J., Grearson, A. G., Willi, J. A., Liu, C. G., Kim, H., Jewett, M., Bellono, N. W., Lee, A. S. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734654</dc:identifier>
<dc:title><![CDATA[Evolution of a core ribosomal innovation in octopus]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734610v1?rss=1">
<title>
<![CDATA[
Expanding the methionine toolkit: L-cyanohomoalanine as a multifunctional analog 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734610v1?rss=1
</link>
<description><![CDATA[
Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.
]]></description>
<dc:creator><![CDATA[ Davis, C. M., Shuster, S. O. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734610</dc:identifier>
<dc:title><![CDATA[Expanding the methionine toolkit: L-cyanohomoalanine as a multifunctional analog]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.26.731617v1?rss=1">
<title>
<![CDATA[
Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.26.731617v1?rss=1
</link>
<description><![CDATA[
Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.
]]></description>
<dc:creator><![CDATA[ Bhuvanendran, H., Brunner, C. M., Kempf, H., Moro, J. L., Roubieu, E., Turbant, F., Mateus, A., Lin, H., Das, L., Malyshev, D., Johns, B., Parracino, A., Pastore, A., Peters, J., Cortajarena, A. L., Zanetti Polzi, L., Maccaferri, N. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.26.731617</dc:identifier>
<dc:title><![CDATA[Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734660v1?rss=1">
<title>
<![CDATA[
Antiglycation effects of imidazole dipeptides and 2-oxo-imidazole dipeptides on glyceraldehyde-induced intracellular protein glycation and neuronal cell death 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734660v1?rss=1
</link>
<description><![CDATA[
Glyceraldehyde (GA) contributes to the development of various diseases, such as diabetes and Alzheimers disease via protein glycation and the formation of advanced glycation end products (AGEs); however, effective strategies for neutralizing GA are limited. Carnosine (Car), an imidazole dipeptide (IDP) that is abundant in meat, suppresses protein glycation by scavenging reactive aldehydes. There are only a few reports on the antiglycation activity of Car against GA. For other IDPs, such as anserine, balenine (Bal), and homocarnosine, there are almost no reports on their antiglycation activity. In this study, we demonstrated the antiglycation activity of four types of IDPs and 2-oxocarnosine (2-oxo-Car), an oxidized form of Car, against GA-induced intracellular protein glycation and neuronal cytotoxicity. Car and Bal exhibited significantly higher reactivity with GA compared with other IDPs and 2-oxo-Car. An in silico analysis suggested that the difference in reactivity is dependent upon intramolecular hydrogen bond formation and the conformation of each IDP. Although there were differences in reactivity with GA, LC-MS analysis revealed that all of the IDPs and 2-oxo-Car reacted with two molecules of GA to form adducts containing pyridinium rings. Car and Bal exhibited high reactivity with GA and markedly suppressed GA-induced cytotoxicity in SH-SY5Y cells. Western blot and qPCR analyses revealed that IDPs suppressed GA-induced protein glycation and the upregulation of endoplasmic reticulum and oxidative stress response genes. Our results indicate that IDPs represent a novel preventive approach to AGE-related diseases and provide a foundation for the development of strategies to treat GA-related neurotoxicity.

Graphical abstractCarnosine and balenine, which are imidazole dipeptides (IDPs), scavenged two molecules of glyceraldehyde to form adducts containing pyridinium rings and suppressed intracellular protein glycation and neuronal cell death. The formation of intramolecular hydrogen bond of IDPs played a crucial role in the strength of antiglycation activity.



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]]></description>
<dc:creator><![CDATA[ Yamada, Y., Hashida, K., Hayashi, K., Yoshimochi, K., Hirose, T., Shimotsuma, M., Hamada, Y., Usui, K., Yokoyama, N., Hara, T., Nishino, S., Kakeya, H., Tomonaga, S., Ozaki, M. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734660</dc:identifier>
<dc:title><![CDATA[Antiglycation effects of imidazole dipeptides and 2-oxo-imidazole dipeptides on glyceraldehyde-induced intracellular protein glycation and neuronal cell death]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734501v1?rss=1">
<title>
<![CDATA[
Hot Pursuit: Bioinformatic and Biochemical Characterization of a Hyperthermophilic Family B DNA Polymerase from Pyrolobus fumarii A1 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734501v1?rss=1
</link>
<description><![CDATA[
Thermostable DNA polymerases are indispensable tools in molecular biology, yet enzymes from the most extreme hyperthermophiles remain largely uncharacterized. Here, we report the biochemical and structural characterization of a family B DNA polymerase from Pyrolobus fumarii A1 (Pyrfu pol), one of the most thermoresistant archaea described to date. The enzyme was efficiently overproduced in E. coli Rosetta 2(DE3)[pLysS] and purified to homogeneity using a two-step protocol that combined heat treatment with immobilized metal affinity chromatography (IMAC). Bioinformatic analysis confirmed the canonical family B architecture, while AlphaFold-based structural modeling and comparative analysis with mesophilic RB69 DNA polymerase revealed a well-conserved structural core alongside thermoadaptive features. Radiolabel incorporation assays demonstrated enzymatic activity over a broad ionic strength range and an absolute requirement for Mg2{square}. PCR-based optimization confirmed these findings and revealed broad pH tolerance (6.5-11.0). Notably, Tris inhibited radiolabel-based assays (pH 7.0) yet proved essential for efficient PCR amplification (pH 8.5), suggesting a context-dependent role of buffer composition in polymerase activity. Processivity assays confirmed amplification of DNA fragments up to approximately 8,000 bp. Replication fidelity, assessed by the lacZ-based assay, showed a 2.9-fold improvement over Taq polymerase. Urea-nanoDSF yielded an exceptional melting temperature of 105.9 {+/-} 0.08 {degrees}C. Pyrfu pol also demonstrated tolerance to common PCR inhibitors, highlighting its potential utility in molecular biology applications.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/734501v1_ufig1.gif" ALT="Figure 1">
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]]></description>
<dc:creator><![CDATA[ Rusinek, W., Dorawa, S., Kaczorowski, T. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734501</dc:identifier>
<dc:title><![CDATA[Hot Pursuit: Bioinformatic and Biochemical Characterization of a Hyperthermophilic Family B DNA Polymerase from Pyrolobus fumarii A1]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.25.734012v1?rss=1">
<title>
<![CDATA[
Small-molecule inhibition of the Orientia tsutsugamushi deubiquitylating enzyme OtDUB impairs bacterial reproduction 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.25.734012v1?rss=1
</link>
<description><![CDATA[
Scrub typhus is a potentially fatal infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. While antibiotic treatment is generally effective, it requires extended treatment, and drug resistance and treatment failures have emerged. O. tsutsugamushi encodes a deubiquitylating enzyme, OtDUB, which interferes with host ubiquitin-dependent pathways. OtDUB cleaves ubiquitin from various substrates, but whether this activity can be selectively targeted by small molecules is unknown. Here we have screened a chemically diverse small-molecule library using a fluorescence-based deubiquitylation assay to identify potential inhibitors of OtDUB. Two compounds, gentisic acid and amiloride hydrochloride, inhibited OtDUB activity at low dosage, with little effect on the related Wolbachia CidB or yeast Ulp1 enzymes. Computational docking predicted the compounds engage regions near the OtDUB catalytic pocket, suggesting a competitive mode of inhibition; this was supported by enzyme kinetic analyses. Neither compound caused detectable cytotoxicity in mammalian cells. Amiloride hydrochloride treatment reduced both total cellular deubiquitylating activity and the O. tsutsugamushi bacterial load in infected cells. While the identified compounds are not optimized inhibitors, they establish that bacterial pathogen-encoded deubiquitylating enzymes can be targeted by small molecules. Overall, our results provide a framework for using selective inhibitors as tools to study DUB function in genetically intractable intracellular bacteria and as potential treatments for scrub typhus.
]]></description>
<dc:creator><![CDATA[ Lee, M. J., Hunt, J. R., Cho, S., Chiarelli, T. J., Perry, C. N., Carlyon, J. A., Hochstrasser, M. ]]></dc:creator>
<dc:date>2026-06-26</dc:date>
<dc:identifier>doi:10.64898/2026.06.25.734012</dc:identifier>
<dc:title><![CDATA[Small-molecule inhibition of the Orientia tsutsugamushi deubiquitylating enzyme OtDUB impairs bacterial reproduction]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-26</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.733881v1?rss=1">
<title>
<![CDATA[
High-Throughput Screening Identifies Small-Molecule Inhibitors of the Tau-LRP1 Interaction 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.733881v1?rss=1
</link>
<description><![CDATA[
The cellular uptake and propagation of tau are central features of tauopathies, including Alzheimers disease, and are mediated by the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1). While prior studies have implicated LRP1 in tau binding and internalization, the biochemical features of this interaction and its suitability for therapeutic targeting remain incompletely defined. Here, we establish a quantitative and scalable framework to interrogate the tau-LRP1 interaction and identify small-molecule modulators. We engineered and purified the LRP1 ligand-binding domain 4 (BD4), a key region mediating tau interaction, and developed multiple orthogonal assays, including fluorescence polarization, split luciferase complementation, and time-resolved FRET, to measure LRP1-BD4 interactions with tau and a known peptide ligand. Across assay formats, we observe consistent binding affinities in the nanomolar range and demonstrate competitive displacement by tau, receptor-associated protein (RAP), and a peptide ligand, supporting overlapping binding interfaces. Leveraging these platforms, we performed small molecule high-throughput screening and identified a set of candidate inhibitors of the LRP1-BD4-tau interaction. Selected compounds reduced tau uptake in a cellular assay, phenocopying competitive inhibition by tau and a peptide ligand. Together, these studies define the LRP1-BD4-tau interaction as a biochemically tractable and druggable interface and establish an integrated discovery pipeline linking mechanistic characterization to functional cellular outcomes. This work provides a foundation for the development of therapeutic strategies targeting LRP1-mediated tau uptake.
]]></description>
<dc:creator><![CDATA[ Wang, C., Ma, C.-T., Crotty, C., Zeng, F.-Y., Bobkov, A., Covel, J. A., Keane Rivera, E., Sergienko, E., Kosik, K. S., Olson, S. H., Jackson, M. R., Rauch, J. N. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.733881</dc:identifier>
<dc:title><![CDATA[High-Throughput Screening Identifies Small-Molecule Inhibitors of the Tau-LRP1 Interaction]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.734323v1?rss=1">
<title>
<![CDATA[
Tracing the Path from 4-Hydroxyphenylpyruvate to the Benzoquinone Ring of Q6 and the p-aminobenzoate pathway in Yeast 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.734323v1?rss=1
</link>
<description><![CDATA[
AbstractCoenzyme Q (ubiquinone, CoQ) is an essential component of the mitochondrial electron transport chain and a major lipid antioxidant in eukaryotic cells. Formation of its benzoquinone ring requires aromatic precursors whose metabolic origin remains incompletely defined. Here, we elucidate the biochemical link between tyrosine metabolism and the synthesis of the benzoquinone head group of coenzyme Q6 (Q6) in Saccharomyces cerevisiae through the 4-hydroxymandelate (4HMA) pathway.

Using isotopic tracing with 13C6-tyrosine, 13C6-4-hydroxybenzoate, and 13C6-p-aminobenzoate (pABA), we demonstrate that tyrosine-derived 4-hydroxyphenylpyruvate is converted into 4-hydroxybenzaldehyde via benzoylformate decarboxylation, defining a functional 4HMA pathway in yeast. Chemical inhibition of benzoylformate decarboxylase with methylbenzoylphosphonate led to accumulation of pathway intermediates, which were identified by GCMS. Consistently, mutants lacking ARO10, DLD1, or DLD2 exhibited strongly decreased 4-hydroxybenzaldehyde formation.

Despite disruption of the 4HMA pathway, the pABA route from chorismate compensated, demonstrating S. cerevisiaes metabolic flexibility to use pABA or 4-HB and maintain Q6 ring biosynthesis. Our results provide a mechanistic framework linking aromatic amino acid metabolism to respiratory quinone biosynthesis in eukaryotes and support the evolutionary conservation of the 4HMA-derived pathway as a source of 4-hydroxybenzoate for Q synthesis in higher organisms.



O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/734323v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1982bddorg.highwire.dtl.DTLVardef@167f888org.highwire.dtl.DTLVardef@ede4eforg.highwire.dtl.DTLVardef@5146fc_HPS_FORMAT_FIGEXP  M_FIG Graphical abstract.4-hydroxyphenylpyruvic acid (4HPP), 4-hydroxymandelate (4HMA), 4-hydroxybenzoic acid (4HB), p-aminobenzoic acid (pABA).

C_FIG
]]></description>
<dc:creator><![CDATA[ Valera Martinez, M. J., Mastrogiovanni, M., Fernandez del Rio, L., Boido, E., Ramos, J. C., Manta, E., Dellacassa, E., Radi, R., Clarke, C. F., Carrau, F. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.734323</dc:identifier>
<dc:title><![CDATA[Tracing the Path from 4-Hydroxyphenylpyruvate to the Benzoquinone Ring of Q6 and the p-aminobenzoate pathway in Yeast]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.734152v1?rss=1">
<title>
<![CDATA[
Self-assembling nanoparticles to assess multivalent interactions between influenza A virus hemagglutinin and glycan surfaces 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.734152v1?rss=1
</link>
<description><![CDATA[
The multivalent display of surface glycoprotein hemagglutinin (HA) on Influenza A viruses (IAVs) enhances the overall binding avidity to sialylated glycans on host cell surfaces. While precomplexing HA trimers with antibodies increases multivalency and avidity, this method does not replicate the virions geometry and limits insights into the multivalent binding process. Here, we use perfectly controllable icosahedral protein nanoparticles to examine the multivalent HA receptor-binding properties. We compare three HA presentation systems with varying degrees of multivalency: single HA trimers, antibody-precomplexed HA trimers, and HA trimers on nanoparticles. Our results indicate that increasing HA valency enhances binding avidity across various glycan surfaces, including erythrocytes, cells, and lipid bilayers with varying glycan densities, while maintaining receptor specificity. By combining functional and non-functional HA trimers during nanoparticle formation, we create statistical mixtures of nanoparticles with varying valencies. At high receptor densities, nanoparticles with few functional trimers still bind strongly, whereas at low receptor densities, a patch of five HA trimers appears necessary for binding. As a key finding, we observe that such a statistical mixture of nanoparticles with functional and nonfunctional HAs binds to glycan surfaces in a stronger density-dependent manner than fully functional particles. We also observe differences in binding modes that correlate with the number of functional trimers, the glycan structure (linear vs branched), and the densities achievable with these glycans. Overall, our findings demonstrate that the presentation of multivalent HA plays an enormous role in the response to glycan receptor type and density, with implications for the future design of virus monitoring, viral inhibitors, and targeting vectors.
]]></description>
<dc:creator><![CDATA[ Rios Carrasco, M., Tambuwun, D. Y. E. L., Ducarne, Z., Turner, H. L., Uslu, E., Ward, A. B., Boons, G.-J., Huskens, J., de Vries, R. P. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.734152</dc:identifier>
<dc:title><![CDATA[Self-assembling nanoparticles to assess multivalent interactions between influenza A virus hemagglutinin and glycan surfaces]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.734353v1?rss=1">
<title>
<![CDATA[
Genetic Code Expansion, Enzymatic Modification, and C-Terminal Labeling Enable Facile Production of Highly Modified α-Synuclein 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.734353v1?rss=1
</link>
<description><![CDATA[
-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.
]]></description>
<dc:creator><![CDATA[ Abakah, B., Shimogawa, M., Miranda-Castrodad, P., Rhoades, E., Petersson, E. J. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.734353</dc:identifier>
<dc:title><![CDATA[Genetic Code Expansion, Enzymatic Modification, and C-Terminal Labeling Enable Facile Production of Highly Modified α-Synuclein]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.734344v1?rss=1">
<title>
<![CDATA[
PARP1 Exhibits an Enzymatically Inactive Chromatin Binding Mode 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.734344v1?rss=1
</link>
<description><![CDATA[
Poly (ADP-ribose) Polymerase 1 (PARP1) is an abundant nuclear enzyme that dynamically engages chromatin in diverse cellular scenarios. In the context of DNA repair, PARP1 becomes enzymatically activated and subsequently attaches ADP-ribose units onto various proteins, including histones, to signal and coordinate the DNA damage response. In the absence of DNA damage, PARP1 modulates chromatin structure by directly binding to nucleosomes, however, the molecular basis of this interaction is unknown. Here, we define a distinct, enzymatically inactive mode of PARP1 chromatin binding, in which the Zn1, Zn2, Zn3, and BRCT domains cooperatively bind nucleosomal linker DNA and drive compaction of undamaged chromatin. This binding mode does not trigger catalytic activation and therefore is insensitive to PARP inhibitors (PARPi). Together, our results support a model in which PARP1 associates with the genome in an inactive state to compact chromatin and to surveil for DNA lesions.

SummaryPARP1 engages undamaged chromatin in a distinct binding mode that results in chromatin compaction but does not lead to enzymatic activation.
]]></description>
<dc:creator><![CDATA[ Fiorenza, A., Anand, M., Luger, K. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.734344</dc:identifier>
<dc:title><![CDATA[PARP1 Exhibits an Enzymatically Inactive Chromatin Binding Mode]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.734275v1?rss=1">
<title>
<![CDATA[
Structural and Biochemical Analysis of the CABIT1 Domain of THEMIS 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.734275v1?rss=1
</link>
<description><![CDATA[
T cells are important components of the adaptive immune system and develop through a selection process regulated by signaling through the T-cell receptor (TCR). Thymocyte-Expressed Molecule Expressed in Selection (THEMIS) is a TCR-proximal protein that modulates the activity of Shp1 phosphatase to influence TCR signaling during development. THEMIS has been shown to both activate and inhibit Shp1, but the molecular mechanisms of these functions are poorly understood. THEMIS contains two rare Cysteine All-Beta In THEMIS (CABIT) domains, the N-terminal of which interacts with Shp1 and is likely responsible for modulation of its phosphatase activity. Herein, we report the first crystal structure of the THEMIS CABIT1 domain. While a portion of the CABIT1 domain is poorly resolved, it appears to share the same overall fold observed in our recent CABIT2 crystal structure and AlphaFold predictions. We show that phosphorylation of the CABIT1 domain by LCK is required for association with SHP1 and that phosphorylated CABIT1 can protect Shp1 from oxidation and inhibition by reactive oxygen species (ROS), which may serve as a mechanism by which THEMIS enhances Shp1 activity.
]]></description>
<dc:creator><![CDATA[ Negron Teron, K. I., Ortiz-Salazar, D., Beyett, T. S. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.734275</dc:identifier>
<dc:title><![CDATA[Structural and Biochemical Analysis of the CABIT1 Domain of THEMIS]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
<item rdf:about="https://www.biorxiv.org/content/10.64898/2026.06.24.734401v1?rss=1">
<title>
<![CDATA[
Regulating Light-Harvesting Protein Assembly through Engineered Trimers of Phycocyanin and Allophycocyanin 
]]>
</title>
<link>
https://www.biorxiv.org/content/10.64898/2026.06.24.734401v1?rss=1
</link>
<description><![CDATA[
Phycobiliproteins form oligomeric assemblies essential for photosynthetic light harvesting. Here, we engineered phycocyanin (TeCPC) and allophycocyanin (TeAPC) from Thermosynechococcus elongatus to stabilize defined trimers by inhibiting hexamer formation. Structure-guided substitutions at conserved glycine residues (TeCPC G29R, TeAPC G21R) introduce steric hindrance at the hexamer interface. Recombinant expression in Escherichia coli produced holoproteins with native-like chromophorylation. Biophysical and structural analyses confirmed homogeneous trimer formation and absence of higher-order assemblies. Thermal measurements indicated cooperative unfolding, supporting structural uniformity. These engineered trimers provide robust models for studying energy transfer in phycobiliproteins.
]]></description>
<dc:creator><![CDATA[ Adachi, M., Tsubouchi, M., Fujita, T., Shibazaki, C., Miyake, K., Itakura, R. ]]></dc:creator>
<dc:date>2026-06-25</dc:date>
<dc:identifier>doi:10.64898/2026.06.24.734401</dc:identifier>
<dc:title><![CDATA[Regulating Light-Harvesting Protein Assembly through Engineered Trimers of Phycocyanin and Allophycocyanin]]></dc:title>
<dc:publisher>Cold Spring Harbor Laboratory</dc:publisher>
<prism:publicationDate>2026-06-25</prism:publicationDate>
<prism:section></prism:section>
</item>
</rdf:RDF>
