Chemical Communications

Transition-metal ion Forster resonance energy transfer (tmFRET) measures short-range distances and conformational changes in proteins, but its distance sensitivity depends on the donor-acceptor pair. Here, we establish the metal-chelating noncanonical amino acid bipyridylalanine (bpyA) as a flexible, genetically encoded acceptor platform whose Forster distance (R0) can be tuned without changing protein labeling. Cu2+, Cu2+ complexed with nitrilotriacetic acid (NTA), and Fe2+ form distinct acceptors at the same bpyA site, allowing distance sensitivity to be adjusted by changing the solution conditions. We incorporated bpyA at two sites in maltose binding protein (MBP) and labeled nearby engineered cysteines with the donor fluorophore BODIPY. The resulting [Cu(bpyA)]2+, [CuNTA(bpyA)]2+, and [Fe(bpyA)]2+ acceptors provided R0 values ranging from 13.9 to 21.2 [A]. Analysis of time-resolved tmFRET data recovered donor-acceptor distance distributions and maltose-dependent changes consistent with structural predictions. At the 237/295 site pair, both copper and iron acceptors resolved the maltose-dependent decrease in distance. At the 309/322 site pair, [Cu(bpyA)]2+ did not appreciably resolve the distance increase, whereas adding NTA shifted R0 toward shorter distances and enabled detection of an increase from 12.1 to 14.4 [A]. These results demonstrate that a single bpyA-labeled protein preparation supports multiple tmFRET acceptors, providing a cysteine-independent acceptor-labeling strategy with adjustable distance sensitivity.

Fluorescent base analogues have proven to be valuable probes of RNA structure. Pyrrolocytosine (pC), an analogue of cytosine, exhibits highly environment-sensitive fluorescence within RNA and is strongly quenched in hydrogen-bonded contexts. In this work, we incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to determine if this quenching mechanism could allow for sensing of RNA-small molecule interactions. We utilized complementary fluorescence spectroscopy methods to under-stand the functional impacts and ligand-sensitivity of pC substitutions distal to and within the binding pocket. We found that pC substitution within a static structural region exhibits the expected ligand-independent fluorescence properties. When placed in the ligand binding pocket, pC exhibits a moderately decreased affinity for the ligands guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. We found that pC behaves as a turn-off sensor, with distinct changes in fluorescence upon G or Hx binding. These differences are dependent on the number of hydrogen bonds formed with pC and are resolvable in both time-dependent and steady-state fluorescence measurements. Our results show that pC can be used to study RNA-small molecule interactions and can discriminate between similar ligands even when they have identical binding modes.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

Reactive metal-oxygen intermediates are central to metalloenzyme catalysis, yet their low populations and short lifetimes make vibrational characterization under aqueous reaction conditions challenging. Here, we establish shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) as a powerful vibrational approach for visualizing reaction-associated metal-ligand signatures of reactive metalloenzyme states. Across the heme and non-heme iron enzymes TauD, VioC, CYP119, and TqaLNc, isotope-sensitive features provide vibrational information on reactive coordination states. In TauD and VioC, isotope-sensitive features provide vibrational characterization of Fe(IV)=O intermediates. In CYP119, an isotope-sensitive Fe(IV)=O vibration is assigned to P450 Compound I generated by m-CPBA, including under lauric-acid turnover conditions. Most notably, in TqaLNc, combined 18O- and 15N-sensitive vibrational responses support formation of the proposed (R)HN-Fe(IV)=O coordination state and the dynamic-coordination mechanism underlying selective C-N bond formation. These results demonstrate that SHINERS can serve as a powerful technique for the in situ vibrational visualization of reactive intermediates and their coordination states in metalloenzyme catalysis.

Plasma membranes (PM) have been traditionally studied by fluorescent probes by either labelling lipid or protein parts. Based on our MemGraft concept, we report a fluorescent probe based on solvatochromic dye Nile Red (MemGraft-NR) for sensing lipid organization in the immediate surrounding of proteins, i.e. at the protein-lipid interface. Two sequentially attached MemGraft scaffolds are required to ensure robust grafting of Nile Red to PM. In contrast to classical lipid-bound membrane probes, MemGraft-NR exhibits high resistance to the presence of serum and fixation with permeabilization. Two-color confocal imaging of MemGraft-NR in live cells reveals higher polarity at the protein-lipid interface compared to lipid-bound probe NR12A. Surprisingly, MemGraft-NR displays higher sensitivity to variation of cholesterol content in PM compared to NR12A, suggesting that protein-lipid interface is particularly impacted by changes in the lipid composition. MemGraft-NR also enabled in situ monitoring of lipid exchange and permeabilization of PM, something impossible for classical solvatochromic PM probes. Unexpectedly, MemGraft-NR reveals a dramatic decrease in the local polarity in early endosomes, suggesting that endosomal negative curvature provides strong impact on organization of the protein-lipid interface. The developed probe opens a new way to study biological membranes by sensing lipid surrounding of membrane proteins.

Neuropeptides regulate a wide range of physiological processes throughout the nervous and endocrine systems, yet their spatiotemporal dynamics in vivo remain poorly understood. While recently developed intensiometric sensors can detect neuropeptides, their use in vivo is often confounded by artifacts induced by hemodynamic changes, pH fluctuations, and motion. Here, we present a general strategy for engineering dual-excitation ratiometric (Ex-ratiometric) neuropeptide sensors by tuning the excited-state proton transfer properties of the fluorescent reporter. Dual excitation at 405 nm and 488 nm produces a ratiometric signal that intrinsically corrects fluorescence fluctuations unrelated to ligand binding. Using an AlphaFold3-guided in silico design approach combined with experimental validation, we developed a suite of Ex-ratiometric sensors. As a representative example, Ex-NTS2.0 enables robust detection of neurotensin (NTS) in vivo while remaining largely resistant to hemodynamics, pH, and motion-induced artifacts. Overall, these findings establish a scalable and mechanistically grounded platform for developing new Ex-ratiometric tools and provide a broadly applicable strategy for sensitive detection of neuropeptide dynamics in living systems.

In the enzymatic toolbox of depollution, hydrolytic dehalogenases are of particular interest for the bioremediation of organohalogenated compounds. To better identify and characterize these enzymes, it is important to develop appropriate sensing methods. Fluorescent probes for dehalogenase detection have been developed in the past years, however, to this date none were available for application using the common FITC parameters (i.e. excitation laser at 488 nm). To address this limitation, we herein report a new fluorescent probe, COU4-Cl, excitable in the violet (405 nm) and blue (488 nm) wavelengths adapted for the detection and characterization of haloalkane dehalogenases. Its applicability is discussed and evaluated in comparison to the previously described COU2-Cl probe only excitable in the violet (405 nm). This study shows the advantages and complementarity of the two probes, and further demonstrate their application for the discrimination of dehalogenase activity in whole cell assays.

Numerous CRISPR-Cas systems have been developed for molecular detection of genetic elements exploiting the trans-cleavage activity of LbCas12a combined with short fluorescent probes. Alongside, a large variability of buffer conditions has been reported. However, how solution chemistry balances enzymatic turnover, signal stability, and detection performance have not been systematically defined. Here, we evaluated how the interplay between anions (chloride vs. acetate), additives, sequence probes, and ionic strength contributes to LbCas12a trans-cleavage activity. Buffer composition screening revealed that chloride-based buffers maintain a low background but slow catalytic rates, meanwhile acetate-based buffers accelerate enzymatic turnover with and without the target DNA sequence, increasing non-specific signals. Reducing agents and surfactants were the best enhancer combinations, improving cleavage performance by increasing ternary complex formation (Km). High salt concentrations only slightly improved the reaction kinetics. Finally, the 5FAM-TTATT-3BHQ-1 reporter showed the best balance between signal intensity and nonspecific background. Our findings indicate that LbCas12a tolerates a vast buffer composition landscape, maintaining robust trans-cleavage activity and detection performance. However, conditions that enhance catalytic activity simultaneously increase nonspecific probe cleavage and higher background, resulting in lower detection performance. Therefore, assay optimization should prioritize detection performance rather than maximal fluorescence output, where solution chemistry is a critical determinant.

Fluorescent proteins (FPs) are the workhorse of modern-day cellular imaging and biosensing. However, rationally predicting and engineering the photophysical properties of FPs remain challenging, as these are governed by complex three-dimensional microenvironments and post-translational modifications. Here, we present a novel surface-mutation-guided engineering strategy combined with iterative rounds of rational mutagenesis to improve key photophysical properties of red fluorescent proteins (RFPs). This approach generated two bright, monomeric RFP variants from the mFruits lineage, mCranberry and mCranberry2, with quantum yields of 85% and 80%, respectively. Strategic incorporation of charged amino acid residues on the protein surface enhanced fluorescence quantum yield without compromising photostability. Comprehensive photophysical characterization and live-cell microscopy demonstrate enhanced performance of these variants and establish their utility as robust probes for fluorescence imaging. However, the exact mechanism of such improvements is subject to further investigation. Collectively, this work introduces a strategy for optimizing fluorescence properties and provides a framework that may be broadly applicable to other pigment-protein systems such as genetically encoded metal-ion indicators and photosynthetic light-harvesting complexes.

We aimed to repurpose the non-nucleoside reverse transcriptase (RT) inhibitor (NNRTI) rilpivirine (RPV) as a targeted protein degrader (TPD) of HIV-1 RT. Structure-guided modeling identified a TPD bifunctional linker exit trajectory from RPV that threads the NNRTI entrance channel. Ten RPV analogs (RPV') with varied warhead-linker attachments retained RT inhibition and antiviral activity, guiding selection of an amide-linked connector for degrader construction. We synthesized Proteolysis Targeting Chimeras (PROTACs) designed to recruit CUL4CRBN and CUL2VHL, alongside adamantyl acetic acid-based Hydrophobic Tags (HyTs). TPDs were evaluated for RT inhibition, virus inhibition, biophysical target engagement, and proteasome-dependent degradation. Some PROTACs showed minimal antiviral activity or limited solubility for our assays, whereas the HyT with a tetraethylene glycol (PEG4) linker (PEG4-Ad) exhibited low nanomolar potency to suppress HIV-1 replication and acceptable solubility. PEG4-Ad reduced RT levels in a proteasome-dependent manner without cytotoxicity but did not exhibit superior potency against RPV resistance mutations. MD simulations suggested that PEG4 linkers maximize episodic exposure of the hydrophobic tag, consistent with PEG4-Ad efficacy. These data highlight the promise - and constraints - of antiviral degraders, indicating that linker-controlled hydrophobic tag exposure and subcellular target accessibility may be critical design parameters for prospective therapeutics.

Polymer modification of antibodies has attracted attention for enhancing antibody functionality. Modification with polyethylene glycol (PEG) or biopolymers such as antibody-oligonucleotide conjugates has been investigated. Although direct effects at the modification site have been understood, distal effects of modifications on intrinsic antibody functions such as antigen-binding and Fc receptor-binding activities remain poorly characterized. In this study, we prepared site-specifically PEGylated trastuzumab to evaluate the effects of the modification site and the size of PEG chains on antibody functions. Reporter gene assays revealed a significant reduction in antibody-dependent cell cytotoxicity (ADCC) activity in a site- and size-dependent manner. Both the reduction in interaction with the antigen and Fc{gamma}RIIIa were observed in a size-dependent manner. Interestingly, the effect of the modification site was only observed for ADCC activity, which suggests that the higher-order structure of the modified antibody plays a critical role in effector function. These results provide molecular design guidelines for the polymer modification of antibodies.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family in the human body and the major targets of FDA-approved medications. Unlike some other physiological proteins, GPCRs are highly plastic and often exist as collections of conformational ensembles in the environment to exert their function. Therefore, understanding how a GPCR adopts different conformational ensembles in response to ligands and signaling partners is essential for drug development. 19F-NMR has emerged as one of the most promising tools in this regard; however, a well-established 19F-labeling system is still lacking, including an effective 19F probe that meets both environmental susceptibility and NMR sensitivity requirements, even though progress has been made dramatically in recent years. Thus, this manuscript reports a novel conformational probe (4-(bromomethyl)-ethoxy-2-(trifluoromethyl)benzene, BTMB) that offers paramount benefits for future GPCR conformational dynamics studies. To rationalize the mechanism behind BTMB's superiority for future probe development, we performed MD simulations, referenced to the current state-of-the-art probe, BTFMA. Our study suggests that the spatial proximity of aromatic residues significantly influences the chemical shift dispersion and linewidths of related 19F-probe-profiled conformational states, as well as the rigidity and compactness of -CF3 in the target protein.

The structural flexibility of RNA is essential for forming ribonucleoprotein (RNP) complexes, which regulate diverse biological processes. This intrinsic property permits RNA to act as a dynamic scaffold along the assembly pathway as it folds into a specific structure for initial recognition by protein and undergoes conformational rearrangements for functional maturation as a complex. Yet, RNA flexibility and RNP multicomponent assembly create significant obstacles for traditional structural methods. To overcome these challenges, we applied recently developed ABEL-FRET spectroscopy to measure tether-free single-molecule Forster resonance energy transfer (smFRET) over extended observation times. Furthermore, ABEL-FRET enables the unique ability for simultaneous measurements of ultrahigh resolution smFRET and hydrodynamic size of individual complexes, which offers distinct advantages for studying dynamic RNA molecules that undergo assembly via sequential binding events. Using ABEL-FRET, we explored how the guide RNA (gRNA) of CRISPR genome editing system folds and modulates its structural flexibility to carry out the roles required for each assembly state from its unbound apo form to the functional Cas9 RNP state for target DNA cleavage. Multi-perspective view of gRNA structure gained by probing its two primary functional domains enabled to capture dramatic changes in gRNA flexibility that are highly dependent on its specific structural domains as well as assembly states. Collectively, our work with ABEL-FRET highlights the intrinsic link between the structural flexibility of RNA and its functionality in RNP assembly.

Proteolysis-targeting chimeras (PROTACs) promote targeted protein degradation by inducing a transient ternary complex between a protein of interest and an E3 ubiquitin ligase. Despite the clinical promise of these event-driven therapeutics, defining the conformational landscape and interfacial organization of these complexes remains challenging. Here, we use an integrated native mass spectrometry (nMS) approach combining ion mobility, surface-induced dissociation (SID), and variable-temperature electrospray ionization (vT-ESI) to determine how small-molecule architecture shapes the structural and thermodynamic landscapes of bromodomain-containing protein 4 (BRD4) with the VCB E3 ligase complex. Using a set of PROTACs that differ in targeting ligand identity (JQ1 versus I-BET726) and PEG linker length, ion mobility measurements show that shorter linkers constrain the ternary complex into more extended topologies, whereas longer, more flexible linkers allow dynamic sampling of relaxed, native-like conformations. SID directly probes noncovalent interfaces and shows that JQ1 complexes favor prompt PROTAC ejection, whereas tighter-binding I-BET726 complexes are more likely to retain the PROTAC at the interface. Complementing these kinetically-controlled gas-phase measurements, vT-ESI reveals a monophasic melting transition for a JQ1-mediated complex and a distinct biphasic melting profile for an I-BET726-mediated complex. The gas-phase activation measurements revealed targeting-ligand-driven differences in PROTAC ejection and retention, whereas vT-ESI uncovered distinct temperature-dependent disassembly pathways for the matched-linker-length JQ10L and IBET10L complexes. Together, these complementary techniques show how PROTAC architecture influences ternary-complex conformation, interfacial connectivity, and disassembly.

Extracellular signal-regulated kinase (ERK) governs many cellular processes, and its spatiotemporal activity dynamics are important for downstream signaling specificity. Existing fluorescent ERK activity biosensors, however, fall short in enabling spectrally multiplexed, spatially resolved, or deep tissue measurements. Here, we introduce a chemigenetic, single-fluorophore biosensor, WHaloEKAR, that reports ERK activities through changes in fluorescence lifetime. WHaloEKAR combines large lifetime changes (-0.81 ns) with the spectral versatility of the self-labeling protein HaloTag giving access to orange, red, and far-red ERK biosensors. The latter enabled the investigation of heterogeneous ERK activity at distinct subcellular locations in cultured cell lines and primary cortical rat neurons via fluorescence lifetime imaging microscopy (FLIM). Moreover, multiplexed FLIM measurements allowed to study ERK activity in parallel to the second messengers calcium and cyclic adenosine monophosphate. Finally, WHaloEKAR enabled the investigation of ERK activity in 3D samples as well as in live animals.

Finite-bandgap charge-based FET biosensors are known to be insensitive to electronic tunneling noise in the semiconductor and interference from weakly-charged proteins in physiological samples. However, their signal is still corrupted by pH fluctuations and condensing counterions in the bulk electrolyte. Here, we demonstrate that electrolyte noise is screened by the Stern layer beyond the linear Debye-Huckel limit, when the interfacial potential drop is larger than the thermal voltage. Using a fully nonlinear Gouy-Chapman-Stern (GCS) theory, a universal calibration curve is derived and shown to collapse the low-noise FET signals for miRNA, extracellular vesicles (EV), and protein, captured either directly or through silica and magnetic nanoparticle charge reporters, over eleven decades of analyte concentration. The theory involves the dissociation constant KD and a gain parameter {chi}, which represents the competing effects of the reporter charge and buffer Debye screening that must be large to produce the low-noise GCS condition. The limit of detection (LOD) is shown to be KD/{chi}, which can be optimized by varying the probe density, charge reporter, and buffer solution. An optimized hybridization buffer then yields {chi} ~ 22 (LOD ~1 pM) for miRNA detection, and multivalent capture reduces KD by four orders of magnitude for EV detection (relative to protein detection) with {chi} ~ 34 (LOD ~10 fM).

Variable-temperature electrospray ionization native mass spectrometry (vT-ESI nMS) provides a means to determine how temperature-dependent changes in protein and solution dynamics influence individual ligand-binding reactions within multiligand protein complexes. Here, the GroEL single-ring mutant (SR1), which binds up to seven nucleotides, serves as a model system due to its sensitivity to solution conditions. Native MS resolves individual ligand-bound populations, while vT-ESI extends these measurements across temperature (3-43 {degrees}C), enabling determination of binding affinities and the associated Gibbs free energy, enthalpy, entropy, heat capacity changes ({Delta}Cp), and enthalpy-entropy compensation (EEC) for each binding reaction. Temperature-dependent changes in van,t Hoff curvature produce distinct {Delta}Cp profiles, indicating changes in the dynamics governing ligand binding that may reflect contributions from protein conformational and protonation microstates. Temperature-dependent shifts in average charge state, consistent with changes in solvent-accessible surface area, indicate corresponding changes in protein conformation. Binding is predominantly enthalpy-driven below ~23 {degrees}C, with increasing entropic contributions at higher temperatures, while the seventh ADP binding reaction exhibits a distinct EEC profile. Comparison of the seventh ADP binding reaction in H2O and D2O reveals pronounced differences in van,t Hoff curvature,{Delta}Cp, and EEC, demonstrating that changes in the hydration environment alter the thermodynamic response. Collectively, these measurements show that temperature-dependent changes in protein and solution dynamics alter the distributions and thermodynamics of individual ligand-bound states and demonstrate the utility of vT-ESI nMS for resolving these effects in complex multiligand systems.

Glycine transporter 1 (GlyT1) is a clinically investigated target whose conformational plasticity enables ligands to engage distinct extracellular and intracellular binding sites. However, methods to directly quantify intracellular binding remain limited and largely rely on radioligand-based approaches. Here, we report a NanoBRET-based binding assay to quantify intracellular GlyT1 target engagement in living cells using a fluorescent tracer derived from the GlyT1 inhibitor Cmpd1. With a structure-based approach followed by pharmacological characterization we identified a suitable meta-substituted exit vector for linker and fluorophore attachment while maintaining GlyT1 binding. A series of fluorescent probes was synthesized and evaluated, leading to the development of GlyT1-probe which displayed high-affinity binding and enabled robust NanoBRET measurements. Unlike the functional uptake assay, the NanoBRET assay distinguished intracellular from extracellular GlyT1 inhibitors and resolved structure-activity relationships at the intracellular binding site. This assay provides a quantitative approach to interrogate conformation-selective GlyT1 target engagement in living cells, facilitating the characterization of GlyT1 ligands targeting the intracellular site.

Catalyst-controlled stereodivergent synthesis of acyclic molecules bearing multiple stereogenic centers remains a longstanding challenge in asymmetric catalysis, particularly through intermolecular radical C-C coupling. Here, we report the directed evolution of pyridoxal 5'-phosphate (PLP)-dependent biocatalysts for the stereodivergent synthesis of non canonical amino acids bearing either skipped , {gamma}-stereochemical dyads or contiguous , {beta}, {gamma}-stereochemical triads. Starting from a thermophilic tryptophan synthase {beta}-subunit, protein engineering generated complementary enzyme variants that selectively construct three of the four possible , {gamma}-stereoisomers through biocatalyst-controlled reversal of both remote {gamma}-stereochemistry and proximal -stereochemistry. These engineered enzymes catalyzed intermolecular radical C-C bond formation with a broad range of secondary alkyltrifluoroborates, affording , {gamma}-disubstituted amino acids in excellent yields and stereoselectivities. Furthermore, directed evolution enabled efficient {beta}-dehydroxylative radical coupling of the non-native threonine-derived aminoacrylate intermediate, providing amino acids bearing contiguous , {beta}, {gamma}-stereochemical triads with up to 96:4:<0.1:<0.1 d.r. and >99:1 e.r.. To further demonstrate the synthetic utility of this platform, the stereochemically enriched amino acids were transformed into valuable {gamma}-chiral amines through engineered PLP dependent tryptophan decarboxylases, establishing a dual PLP enzyme based biocatalytic cascade for the enantiodivergent synthesis of privileged amine scaffolds. Molecular dynamics simulations reveal that enzyme controlled {gamma}-stereoselectivity originates from active-site remodeling that modulates {pi}-{pi} interactions and steric contacts governing the facial selectivity of the radical addition step. This work expands the synthetic capabilities of radical biocatalysis by enabling catalyst-controlled stereodivergent assembly of complex acyclic stereochemical arrays and establishes engineered PLP enzymes as versatile catalysts for the synthesis of stereochemically complex amino acids and chiral amines.

Antibody-oligonucleotide conjugates (AOCs) offer a promising solution to delivery challenges of therapeutic oligonucleotides. However, the relationship between their complex chemical architectures and biological activity remains poorly understood, limiting the development of important structure-function relationships. For siRNA-containing AOCs, increasing the siRNA-to-antibody ratio beyond one (drug-to-antibody ratio, DAR>1) reduces potency, attributed to altered pharmacokinetics arising from increased negative charge density. Here, we investigated whether simple chemical modifications could improve AOC efficacy and mitigate limitations associated with higher DAR. Introduction of a single C16 lipid modification to the siRNA significantly enhanced target gene silencing compared to the unmodified AOC. Extending this modification to a DAR2 architecture, in which two siRNAs are conjugated per antibody, restored the loss of activity associated with increasing DAR from 1 to 2. Notably, at an equivalent siRNA dose (1 mg/kg), the DAR2-C16 AOC requires half the amount of antibody while achieving knockdown comparable to the DAR1-C16 AOC. We also developed a charge-balancing ionizable linker (CBIL) designed to partially compensate for the negative charge of siRNA. Incorporation of the CBIL enhanced target gene silencing in heart and skeletal muscle without a corresponding increase in hepatic activity, resulting in a shift toward greater extrahepatic activity relative to liver. Together, these findings demonstrate that chemical modification of both the siRNA payload and antibody-siRNA linker can be used to tune AOC potency and tissue activity, while enabling higher payload loading without compromising efficacy. These results establish chemical design as an important strategy for expanding the architecture and therapeutic potential of AOCs.