HaloTag (HT) is a versatile self-labeling protein that has been widely adopted in fluorescence microscopy. Besides its established use as an intensity-based marker and sensor, interest in using HT for multiplexed fluorescence lifetime imaging microscopy (FLIM) has recently arisen. Herein, the application of an environment-sensitive fluorophore was explored for FLIM multiplexing with the free dye and HT mutants. The extended coumarin pyridinium scaffold was selected due to its structural simplicity and the strong sensitivity of its photophysical properties to the environment. It was demonstrated that three-channel imaging is possible by taking advantage of the propensity of the dye to accumulate in mitochondria and vesicles and the efficient labeling of two distinct HT mutants. Further investigation was conducted on different dehalogenase proteins and their FLIM multiplexing capabilities when paired with an HT mutant. Finally, the polarity of the protein binding pocket was identified as a key feature that affects the lifetime of this kind of fluorescent molecule.

Different from reversible agonist-stimulated receptor activation, singlet oxygen oxidation activates permanently G protein-coupled receptor (GPCR) cholecystokinin 1 (CCK1R) in type II photodynamic action, with soluble photosensitizer dyes (sulphonated aluminum phthalocyanine, λmax 675 nm) or genetically encoded protein photosensitizers (KillerRed λmax 585 nm; mini singlet oxygen generator λmax 450 nm), together with a pulse of light (37 mW/cm2, 1-2 minutes). Three lines of evidence shed light on the mechanism of GPCR activated by singlet oxygen (GPCR-ABSO): (1) CCK1R is quantitatively converted from dimer to monomer; (2) Transmembrane domain 3, a pharmacophore for permanent photodynamic CCK1R activation, can be transplanted to non-susceptible M3 acetylcholine receptor; and (3) Larger size of disordered region in intracellular loop 3 correlates with higher sensitivity to photodynamic CCK1R activation. GPCR-ABSO will add to the arsenal of engineered designer GPCR such as receptors activated solely by synthetic ligands and designer receptors exclusively activated by designer drugs, but show some clear advantages: Enhanced selectivity (double selectivity of localized photosensitizer and light illumination), long-lasting activation with no need for repeated drug administration, antagonist-binding site remains intact when needed, ease to apply to multiple GPCR. This type of permanent photodynamic activation may be applied to functional proteins other than GPCR.

Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal-length DNAs and mRNAs to improve molecular display principle for target identification. The equal-length DNAs were designed to contain various coding sequences for full-length proteins with molecular weights of up to 130 kDa and were used to synthesize equal-length mRNAs, which allowed the formation of full-length protein-ribosome-equal-length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein-displaying ribosomal complexes were specifically selected through non-covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full-length proteins interacting with methotrexate or anti-DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal-length mRNAs encoding them were adjusted to 1/20,000 of the total equal-length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.

Protein hydration plays a vital role in many biological functions, and molecular dynamics simulations are frequently used to study it. However, the accuracy of these simulations is often sensitive to the water model used, a phenomenon particularly evident in intrinsically disordered proteins. Here, we investigated the extent to which the choice of water model alters the behavior of complex networks of tunnels within proteins. Tunnels are essential because they allow the exchange of substrates and products between buried enzyme active sites and the bulk solvent, directly affecting enzyme efficiency and selectivity, making the study of tunnels crucial for a holistic understanding of enzyme function at the molecular level. By performing simulations of haloalkane dehalogenase LinB and its two variants with engineered tunnels using TIP3P and OPC models, we investigated their effects on the overall tunnel topology. We also analyzed the properties of the primary tunnels, including their conformation, bottleneck dimensions, sampling efficiency, and the duration of tunnel openings. Our data demonstrate that all three proteins exhibited similar conformational behavior in both models but differed in the geometrical characteristics of their auxiliary tunnels, consistent with experimental observations. Interestingly, the results indicate that the stability of the open tunnels might be sensitive to the water model used. Because TIP3P can provide comparable data on the overall tunnel network, it is a valid choice when computational resources are limited or compatibility issues impede the use of OPC. However, OPC seems preferable for calculations requiring an accurate description of transport kinetics.

Self-labeling proteins are powerful tools for exploring biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye. HaloTag7 is the most popular self-labeling protein due to its broad utility, its bio-orthogonality, and the simplicity of its chloroalkane ligand. However, reaction rates of HaloTag7 with different chloroalkane-containing substrates are highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we use yeast surface display to produce a HaloTag variant, BenzoTag, with improved performance with a fluorogenic benzothiadiazole dye. Molecular evolution improved conjugation kinetics and increased the signal from the dye-protein complex, allowing for robust, no-wash fluorescence labeling in live cells. The new BenzoTag-benzothiadiazole system has improved performance compared to the best existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. The BenzoTag system was also found to be sufficiently orthogonal to the HaloTag7-silicon rhodamine system to enable multiplexed no-wash labeling in live cells. The BenzoTag system will be immediately useful for a large variety of cell-based assays monitoring biological processes and drug action in real time.

Immobilized proteins hold promise as the basic units that have enabled a broad range of analytical applications within chemical measurement science. As yet, the co-immobilization of diverse proteins at precise ratio and whether they give rise to improved analytical performance remain challengeable. Herein, we utilized a circularly permuted HaloTag (cpHaloTag) to achieve the co-immobilization of two proteins at precise ratio, which was applied in developing a chromatographic method with improved specificity for pursuing dual-target compounds.

The methodology involved the fusion 3A and 2C at N- and C-terminuses of cpHaloTag, the immobilization of the fusion protein onto silica gel through bioorthogonal reaction, the morphological and functional characterization, the application in finding dual-target compounds. Expression of the fusion protein in E. coli system showed a yield of milligram level with the presence of 3A and 2C domains. Immobilization of the protein was achieved in 10 min with a reaction efficiency more than 88.5 %. Immobilized 3A-cpHalo-2C exhibited higher specificity and better retentions of canonical compounds of the two enzymes in comparison with the column containing immobilized 3A or 2C alone. In real sample application, screening analysis found that hyperoside, cymaroside, and baicalin were dual-target compounds in concert with 3A and 2C in Shuanghuanglian extract.

Taking 3A and 2C as probe, we proposed a simple method for direct co-immobilization of diverse proteins from cell lysates and demonstrated an affinity chromatographic-based dual-target compound screening platform. The implications of these methodology are possible to insight the de novo design of multi-target surface for fabricating new bioanalytical methods with improved performance.

The combination of fluorogenic probes (fluorogens) and self-labeling protein tags represent a promising tool for imaging biological processes with high specificity but it requires the adequation between the fluorogen and its target to ensure a good activation of its fluorescence. In this work, we report a strategy to develop molecular rotors that specifically target HaloTag with a strong enhancement of their fluorescence. The divergent design facilitates the diversification of the structures to tune the photophysical and cellular properties. Four bright fluorogens with emissions ranging from green to red were identified and applied in wash-free live cell imaging experiments with good contrast and selectivity. A HaloTag mutant adapted from previous literature reports was also tested and shown to further improve the brightness and reaction rate of the most promising fluorogen of the series both in vitro and in cells. This work opens new possibilities to develop bright chemogenetic reporters with diverse photophysical and biological properties by exploring a potentially large chemical space of simple dipolar fluorophores in combination with protein engineering.

Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for in vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to rapidly detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.

Self-labeling proteins are powerful tools in chemical biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye, with the genetic specificity of a protein fusion. HaloTag7 is the most popular self-labeling protein due to its fast labeling kinetics and the simplicity of its chloroalkane ligand. Reaction rates of HaloTag7 with different chloroalkane-containing substrates is highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we report a molecular evolution system for HaloTag using yeast surface display that enables the screening of libraries up to 108 variants to improve reaction rates with any substrate of interest. We applied this method to produce a HaloTag variant, BenzoHTag, which has improved performance with a fluorogenic benzothiadiazole dye. The resulting system has improved brightness and conjugation kinetics, allowing for robust, no-wash fluorescent labeling in live cells. The new BenzoHTag-benzothiadiazole system has improved performance in live-cell assays compared to the existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. It was also found to be orthogonal to the silicon HaloTag7-rhodamine system, enabling multiplexed no-wash labeling in live cells. The BenzoHTag system, and the ability to optimize HaloTag for a broader collection of substrates using molecular evolution, will be very useful for the development of cell-based assays for chemical biology and drug development.

Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

Post-translational modifications (PTMs) are crucial mechanisms that underlie the intricacies of biological systems and disease mechanisms. This review focuses on the latest advancements in the design of heterobifunctional small molecules that hijack PTM machineries for target-specific modifications in living systems. A key innovation in this field is the development of proteolysis-targeting chimeras (PROTACs), which promote the ubiquitination of target proteins for proteasomal degradation. The past decade has seen several adaptations of the PROTAC concept to facilitate targeted (de)phosphorylation and acetylation. Protein fusion tags have been particularly vital in these proof-of-concept studies, aiding in the investigation of the functional roles of post-translationally modified proteins linked to diseases. This overview delves into protein-tagging strategies that enable the targeted modulation of ubiquitination, phosphorylation, and acetylation, emphasizing the synergies and challenges of integrating heterobifunctional molecules with protein tags in PTM research. Despite significant progress, many PTMs remain to be explored, and protein tag-assisted PTM-inducing chimeras will continue to play an important role in understanding the fundamental roles of protein PTMs and in exploring the therapeutic potential of manipulating protein modifications, particularly for targets not yet addressed by existing drugs.

Mass cytometry permits the high dimensional analysis of complex biological samples; however, some techniques are not yet integrated into the mass cytometry workflow due to reagent availability. The use of self-labeling protein systems, such as HaloTag, are one such application. Here, we describe the design and implementation of the first mass cytometry ligands for use with HaloTag. "Click"-amenable HaloTag warheads were first conjugated onto poly(l-lysine) or poly(acrylic acid) polymers that were then functionalized with diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates. Kinetic analysis of the HaloTag labeling rates demonstrated that the structure appended to the 1-chlorohexyl warhead was key to success. A construct with a diethylene glycol spacer appended to a benzamide gave similar rates (kobs ∼ 102 M-1 s-1), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics (kobs ∼ 104 M-1 s-1) suggests the polymers significantly reduced the HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag fusions were labeled with the polymeric constructs and 175Lu content measured by cytometry by time-of-flight (CyTOF). Robust labeling was observed; however, significant nonspecific binding of the constructs to cells was also present. Heavily pegylated polymers demonstrated that nonspecific binding could be reduced to allow cells bearing the HaloTag protein to be distinguished from nonexpressing cells.

Recent years have seen dramatic improvements in the design of organic fluorophores based on limiting non-radiative decay pathways. We sought to extend this understanding to benzothiadiazoles that have been used as turn-on fluorescent substrates for the self-labeling protein HaloTag. When conjugated to HaloTag, the benzothiadiazoles reside in a narrow tunnel that precludes twisted internal charge transfer, which allowed us to explore steric and electronic effects on other non-radiative decay pathways. By minimizing both non-radiative decay and nonspecific interactions with cellular components, we produced improved turn-on dyes with 136-fold increase in fluorescence over background in cells.

Cell membrane coating strategies have been increasingly researched due to their unique capabilities of biomimicry and biointerfacing, which can mimic the functionality of the original source cells in vivo but fail to provide customized nanoparticle surfaces with new or enhanced capabilities beyond natural cells. However, the field of drug lead discovery necessitates the acquisition of sufficient surface density of specific target membrane receptors, presenting a heightened demand for this technology. In this study, we developed a novel approach to fabricate high density of fibroblast growth factor receptor 4 (FGFR4) cell membrane-coated nanoparticles through covalent site-specific immobilization between genetically engineered FGFR4 with HaloTag anchor on cell membrane and chloroalkane-functionalized magnetic nanoparticles. This technique enables efficient screening of tyrosine kinase inhibitors from natural products. And the enhanced density of FGFR4 on the surface of nanoparticles were successfully confirmed by Western blot assay and confocal laser scanning microscopy. Further, the customized nanoparticles demonstrated exceptional sensitivity (limit of detection = 0.3 × 10-3 μg mL-1). Overall, the proposed design of a high density of membrane receptors, achieved through covalent site-specific immobilization with a HaloTag anchor, demonstrates a promising strategy for the development of cell membrane surface engineering. This approach highlights the potential of cell membrane coating technology for facilitating the advanced extraction of small molecules for drug discovery.

Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

Photoactivatable fluorescence imaging is one of the most valuable methods for visualizing protein localization, trafficking, and interactions. Here, we designed four bioorthogonal fluorescent probes K1-K4 by installing photoactive cages and HaloTag ligands onto the different positions of the coumarin fluorophore. Although K1-K4 all exhibited rapid photostimulated responses in aqueous solution, only K3 was found to have an obvious aggregation-induced emission (AIE). Next, macromolecular fluorescent probes Kn=1/2/3/4_POIs were obtained by covalently attaching K1-K4 to HaloTag-fused proteins of interest (POIs). Kn=3/4_POIs exhibited a higher fluorescence increase than that of Kn=1/2_POIs upon photoactivation in both liquid and solid phases. Moreover, K3_GFP_Halo and K4_GFP_Halo presented the fluorescence resonance energy transfer (FRET) from photocleaved K3 and K4 to GFP in the protein complex. We further examined the fluorescence labeling ability of K1-K4 to intracellular IRE1_Halo protein and found that K3 and K4 containing the HaloTag ligand on the C4 position of coumarin could be retained in cells for long-term tracking of the IRE1_Halo protein. Hence, we established a platform of novel bioorthogonal fluorescent probes conjugating onto Halo-tagged POIs for rapid photoactivation in vitro and in cells.