Conditional requirement for dimerization of the membrane-binding module for BTK signaling in lymphocyte cell lines.

Bruton's tyrosine kinase (BTK) is a major drug target in immune cells. The membrane-binding pleckstrin homology and tec homology (PH-TH) domains of BTK are required for signaling. Dimerization of the PH-TH module strongly stimulates the kinase activity of BTK in vitro.

Electron transfer in polysaccharide monooxygenase catalysis.

Polysaccharide monooxygenase (PMO) catalysis involves the chemically difficult hydroxylation of unactivated C-H bonds in carbohydrates. The reaction requires reducing equivalents and will utilize either oxygen or hydrogen peroxide as a cosubstrate. Two key mechanistic questions are addressed here: 1) How does the enzyme regulate the timely and tightly controlled electron delivery to the mononuclear copper active site, especially when bound substrate occludes the active site? and 2) How does this electron delivery differ when utilizing oxygen or hydrogen peroxide as a cosubstrate? Using a computational approach, potential paths of electron transfer (ET) to the active site copper ion were identified in a representative AA9 family PMO from (PMO9E).

Mechanism-guided engineering of a minimal biological particle for genome editing.

The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization.

Wireless power-up and readout from a label-free biosensor.

Wearable and implantable biosensors have rapidly entered the fields of health and biomedicine to diagnose diseases and physiological monitoring. The use of wired medical devices causes surgical complications, which can occur when wires break, become infected, generate electrical noise, and are incompatible with implantable applications. In contrast, wireless power transfer is ideal for biosensing applications since it does not necessitate direct connections between measurement tools and sensing systems, enabling remote use of the biosensors.

Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications.

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 on K27 (H3K27me3) leading to gene silencing that is essential for embryonic development and maintenance of cell identity. PRC2 is regulated by protein cofactors and their crosstalk with histone modifications. Trimethylated histone H3 on K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription and inhibit PRC2 activity through unknown mechanisms.

AI-Based Prediction of Protein Corona Composition on DNA Nanostructures.

DNA nanotechnology has emerged as a powerful approach to engineering biophysical tools, therapeutics, and diagnostics because it enables the construction of designer nanoscale structures with high programmability. Based on DNA base pairing rules, nanostructure size, shape, surface functionality, and structural reconfiguration can be programmed with a degree of spatial, temporal, and energetic precision that is difficult to achieve with other methods. However, the properties and structure of DNA constructs are greatly altered due to spontaneous protein adsorption from biofluids.

Recombinant Expression of Photo-crosslinkable 26S Proteasome Base Subcomplex.

The 26S proteasome complex is the hub for regulated protein degradation in the cell. It is composed of two biochemically distinct complexes: the 20S core particle with proteolytic active sites in an internal chamber and the 19S regulatory particle, consisting of a lid and base subcomplex. The base contains ubiquitin receptors and an AAA+ (ATPases associated with various cellular activities) motor that unfolds substrates prior to degradation.

Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in .

The integrated stress response (ISR) is a conserved eukaryotic signaling pathway that responds to diverse stress stimuli to restore proteostasis. The strength and speed of ISR activation must be tuned properly to allow protein synthesis while maintaining proteostasis. Here, we describe how genetic perturbations change the dynamics of the ISR in budding yeast.

Direct and quantitative analysis of tRNA acylation using intact tRNA liquid chromatography-mass spectrometry.

Aminoacyl-tRNA synthetases (aaRSs) provide an essential functional link between an mRNA sequence and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases.

Functional protein mining with conformal guarantees.

Molecular structure prediction and homology detection offer promising paths to discovering protein function and evolutionary relationships. However, current approaches lack statistical reliability assurances, limiting their practical utility for selecting proteins for further experimental and in-silico characterization. To address this challenge, we introduce a statistically principled approach to protein search leveraging principles from conformal prediction, offering a framework that ensures statistical guarantees with user-specified risk and provides calibrated probabilities (rather than raw ML scores) for any protein search model.

Best Practices and Pitfalls in Developing Nanomaterial Delivery Tools for Plants.

Numerous reports of nanomaterial-assisted delivery of DNA, RNA, and protein to plants for biotechnology applications emerged over the past decade. While the field has experienced rapid growth, best practices for developing and validating nanomaterial delivery tools for plants have not yet been established. Best practices are well-established for clinical/animal cell delivery experiments, yet plants pose a distinct challenge requiring separate considerations due to their unique tissue structures and cellular morphology.

Fe starvation induces a second LHCI tetramer to photosystem I in green algae.

Iron (Fe) availability limits photosynthesis at a global scale where Fe-rich photosystem (PS) I abundance is drastically reduced in Fe-poor environments. We used single-particle cryo-electron microscopy to reveal a unique Fe starvation-dependent arrangement of light-harvesting chlorophyll (LHC) proteins where Fe starvation-induced TIDI1 is found in an additional tetramer of LHC proteins associated with PSI in and . These cosmopolitan green algae are resilient to poor Fe nutrition.

Dual Infrared 2-Photon Microscopy Achieves Minimal Background Deep Tissue Imaging in Brain and Plant Tissues.

Traditional deep fluorescence imaging has primarily focused on red-shifting imaging wavelengths into the near-infrared (NIR) windows or implementation of multi-photon excitation approaches. Here, we combine the advantages of NIR and multiphoton imaging by developing a dual-infrared two-photon microscope to enable high-resolution deep imaging in biological tissues. We first computationally identify that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation.

CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.

RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes.

Covalent Attachment of Horseradish Peroxidase to Single-Walled Carbon Nanotubes for Hydrogen Peroxide Detection.

Single-walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near-infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre-existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging.

Analyses of translation factors Dbp1 and Ded1 reveal the cellular response to heat stress to be separable from stress granule formation.

Ded1 and Dbp1 are paralogous conserved DEAD-box ATPases involved in translation initiation in yeast. In long-term starvation states, Dbp1 expression increases and Ded1 decreases, whereas in cycling mitotic cells, Dbp1 is absent. Inserting DBP1 in place of DED1 cannot replace Ded1 function in supporting mitotic translation, partly due to inefficient translation of the DBP1 coding region.

Reactive oxygen species control protein degradation at the mitochondrial import gate.

While reactive oxygen species (ROS) have long been known to drive aging and neurodegeneration, their persistent depletion below basal levels also disrupts organismal function. Cells counteract loss of basal ROS via the reductive stress response, but the identity and biochemical activity of ROS sensed by this pathway remain unknown. Here, we show that the central enzyme of the reductive stress response, the E3 ligase Cullin 2-FEM1 homolog B (CUL2), specifically acts at mitochondrial TOM complexes, where it senses ROS produced by complex III of the electron transport chain (ETC).

RNA language models predict mutations that improve RNA function.

Structured RNA lies at the heart of many central biological processes, from gene expression to catalysis. RNA structure prediction is not yet possible due to a lack of high-quality reference data associated with organismal phenotypes that could inform RNA function. We present GARNET (Gtdb Acquired RNa with Environmental Temperatures), a new database for RNA structural and functional analysis anchored to the Genome Taxonomy Database (GTDB).

Structural basis for growth factor and nutrient signal integration on the lysosomal membrane by mTORC1.

Mechanistic target of rapamycin complex 1 (mTORC1), which consists of mTOR, Raptor, and mLST8, receives signaling inputs from growth factor signals and nutrients. These signals are mediated by the Rheb and Rag small GTPases, respectively, which activate mTORC1 on the cytosolic face of the lysosome membrane. We biochemically reconstituted the activation of mTORC1 on membranes by physiological submicromolar concentrations of Rheb, Rags, and Ragulator.

UPR deficiency in budding yeast reveals a trade-off between ER folding capacity and maintenance of euploidy.

The Unfolded Protein Response (UPR) was discovered in budding yeast as a mechanism that allows cells to adapt to ER stress. While the Ire1 branch of this pathway is highly conserved, it is not thought to be important for cellular homeostasis in the absence of stress. Surprisingly, we found that removal of UPR activity led to pervasive aneuploidy in budding yeast cells, suggesting selective pressure resulting from UPR-deficiency.

Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second strand nicking.

R2 retrotransposons are model site-specific eukaryotic non-LTR retrotransposons that copy-and-paste into gene loci encoding ribosomal RNAs. Recently we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not characterized.

Asynchronous abundance fluctuations can drive giant genotype frequency fluctuations.

Large stochastic population abundance fluctuations are ubiquitous across the tree of life, impacting the predictability and outcomes of population dynamics. It is generally thought that abundance fluctuations with a Taylor's law exponent of two do not strongly impact evolution. However, we argue that such abundance fluctuations can lead to substantial genotype frequency fluctuations if different genotypes in a population experience these fluctuations asynchronously.