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The transcription factor NF-ĸB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-ĸB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-ĸB/IĸBα/DNA interaction network. The development of genetic incorporation of non-canonical amino acids (ncAA) has enabled the installation of biophysical probes into proteins with site specificity. Recent single-molecule FRET (smFRET) studies of NF-ĸB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IĸBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-ĸB and site-specific fluorophore labeling with copper-free click chemistry for smFRET. We also expanded the ncAA toolbox of NF-ĸB to include p-benzoylphenylalanine (pBpa) for UV crosslinking mass spectrometry (XL-MS) and incorporated both pAzF and pBpa into the full-length NF-ĸB RelA subunit which includes the intrinsically disordered transactivation domain.

• XPA is an essential protein for the repair of bulky DNA lesions through Nucleotide Excision Repair. • XPA works in complex with RPA to orient the DNA for processing. • A site-specific Cy3 fluorophore was incorporated on XPA and reports on ssDNA binding dynamics. • Detailed methods to generate fluorescent XPA and conditions to generate and maintain stable protein are described. Nucleotide excision repair (NER) promotes genomic integrity by removing bulky DNA adducts introduced by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the ss/ds DNA junction. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido- L -phenylalanine (4AZP or pAzF) was incorporated at Arg-158 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent XPA protein (XPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate XPACy3 and detail in vitro experimental conditions required to stably maintain the protein during biochemical and biophysical studies. [ABSTRACT FROM AUTHOR]

• Site-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics. • Bulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA. • Experiments capture the dynamic ssDNA binding states of the DBD-A and DBD-D domains of human RPA. DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido- L- phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions. [ABSTRACT FROM AUTHOR]

Nanobodies (Nbs) hold significant potential in molecular imaging due to their unique characteristics. However, there are challenges to overcome when it comes to brain imaging. To address these obstacles, collaborative efforts and interdisciplinary research are needed. This article aims to raise awareness and encourage collaboration among researchers from various fields to find solutions for effective brain imaging using Nbs. By fostering cooperation and knowledge sharing, we can make progress in overcoming the existing limitations and pave the way for improved molecular imaging techniques in the future. We outline in this article the advantages and challenges associated with utilizing nanobodies for brain imaging. As this field is still in its nascent stages, we encourage scientists to share their discoveries related to blood‐brain barrier‐penetrating Nbs generously. Together, we would like to collectively address the obstacles and pave the way for more effective brain receptor imaging in the near future. [ABSTRACT FROM AUTHOR]

Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spectrometry (CL-MS) to assess structural changes in a model mAb (SILuMAb). Structural changes in the heat-stressed mAb samples were confirmed at specific amino acid positions from the DEPC label mass seen in the fragment ion mass spectrum. The degree of structural change was also quantified by increased or decreased DEPC labeling at specific sites; an increase or decrease indicated an unfolded or aggregated state of the mAb, respectively. Strikingly, for heat-stressed SILuMAb samples, an aggregation-prone area was identified in the CDR region. In the case of longterm stress, the structural consequences for SILuMAb samples stored for up to two years at 2–8 °C were studied with SEC-UV and DEPC-based CL-MS. While SEC-UV analysis only indicated fragmentation of SILuMAb, DEPC-based CL-MS analysis further pinpointed the finding to structural disturbances of disulfide bonds at specific cysteines. This emphasized the utility of DEPC CL-MS for studying disulfide rearrangement. Taken together, our data suggests that DEPC CL-MS can complement more technically challenging methods in the evaluation of the structural stability of mAbs. [ABSTRACT FROM AUTHOR]

Despite advances in immunotherapy for the treatment of cancers, not all patients can benefit from programmed cell death ligand 1 (PD-L1) immune checkpoint blockade therapy. Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level; hence, accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects. Therefore, based on the high affinity antibody Nb109, a new site-specifically radiolabeled tracer, 68Ga-NODA-cysteine, aspartic acid, and valine (CDV)-Nb109, was designed and synthesized to accurately monitor PD-L1 expression. The tracer 68Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95% and radiochemical purity of 97%. It showed high affinity for PD-L1 with a dissociation constant of 12.34 ± 1.65 nM. Both the cell uptake assay and positron emission tomography (PET) imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells. Meanwhile, dynamic PET imaging of a NCI-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression, allowing timely interventional immunotherapy. In conclusion, this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy.

• Monitoring of human SBDS activity using noncanonical amino acid labeling. • Select domains of SBDS can be site-specifically labeled. • Fluorescence anisotropy captures interactions between labeled SBDS and 60S subunit. The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits. [ABSTRACT FROM AUTHOR]

Studies of structural changes in mAbs under forced stress and storage conditions are essential for the recognition of degradation hotspots, which can be further remodeled to improve the stability of the respective protein. Herein, we used diethyl pyrocarbonate (DEPC)-based covalent labeling mass spectrometry (CL-MS) to assess structural changes in a model mAb (SILuMAb). Structural changes in the heat-stressed mAb samples were confirmed at specific amino acid positions from the DEPC label mass seen in the fragment ion mass spectrum. The degree of structural change was also quantified by increased or decreased DEPC labeling at specific sites; an increase or decrease indicated an unfolded or aggregated state of the mAb, respectively. Strikingly, for heat-stressed SILuMAb samples, an aggregation-prone area was identified in the CDR region. In the case of longterm stress, the structural consequences for SILuMAb samples stored for up to two years at 2–8 °C were studied with SEC-UV and DEPC-based CL-MS. While SEC-UV analysis only indicated fragmentation of SILuMAb, DEPC-based CL-MS analysis further pinpointed the finding to structural disturbances of disulfide bonds at specific cysteines. This emphasized the utility of DEPC CL-MS for studying disulfide rearrangement. Taken together, our data suggests that DEPC CL-MS can complement more technically challenging methods in the evaluation of the structural stability of mAbs.

The knowledge of interactions among functional proteins helps researchers understand disease mechanisms and design potential strategies for treatment. As a general approach, the fluorescent and affinity tags were employed for exploring this field by labeling the Protein of Interest (POI). However, the autofluorescence and weak binding strength significantly reduce the accuracy and specificity of these tags. Conversely, HaloTag, a novel self-labeling enzyme (SLE) tag, could quickly form a covalent bond with its ligand, enabling fast and specific labeling of POI. These desirable features greatly increase the accuracy and specificity, making the HaloTag a valuable system for various applications ranging from imaging to immobilization of POI. Notably, the HaloTag technique has already been successfully employed in a series of studies with excellent efficiency. In this review, we summarize the development of HaloTag and recent advanced investigations associated with HaloTag, including in vitro imaging (e.g., POI imaging, cellular condition monitoring, microorganism imaging, system development), in vivo imaging, biomolecule immobilization (e.g., POI collection, protein/nuclear acid interaction and protein structure analysis), targeted degradation (e.g., L-AdPROM), and more. We also present a systematic discussion regarding the future direction and challenges of the HaloTag technique. [ABSTRACT FROM AUTHOR]

Despite advances in immunotherapy for the treatment of cancers, not all patients can benefit from programmed cell death ligand 1 (PD-L1) immune checkpoint blockade therapy. Anti-PD-L1 therapeutic effects reportedly correlate with the PD-L1 expression level; hence, accurate detection of PD-L1 expression can guide immunotherapy to achieve better therapeutic effects. Therefore, based on the high affinity antibody Nb109, a new site-specifically radiolabeled tracer, 68Ga-NODA-cysteine, aspartic acid, and valine (CDV)-Nb109, was designed and synthesized to accurately monitor PD-L1 expression. The tracer 68Ga-NODA-CDV-Nb109 was obtained using a site-specific conjugation strategy with a radiochemical yield of about 95% and radiochemical purity of 97%. It showed high affinity for PD-L1 with a dissociation constant of 12.34 ± 1.65 nM. Both the cell uptake assay and positron emission tomography (PET) imaging revealed higher tracer uptake in PD-L1-positive A375-hPD-L1 and U87 tumor cells than in PD-L1-negative A375 tumor cells. Meanwhile, dynamic PET imaging of a NCI-H1299 xenograft indicated that doxorubicin could upregulate PD-L1 expression, allowing timely interventional immunotherapy. In conclusion, this tracer could sensitively and dynamically monitor changes in PD-L1 expression levels in different cancers and help screen patients who can benefit from anti-PD-L1 immunotherapy. [Display omitted] • Development of a radiolabeled site-specific single-domain antibody PET probe. • High affinity to PD-L1 in vitro and in vivo. • This tracer can noninvasively report PD-L1 expression in different tumors. • Changes in PD-L1 expression induced by chemotherapy can be sensitively detected. [ABSTRACT FROM AUTHOR]

Super-resolution microscopy (SRM) has transformed our understanding of proteins' subcellular organization and revealed cellular details down to nanometers, far beyond conventional microscopy. While localization precision is independent of the number of fluorophores attached to a biomolecule, labeling density is a decisive factor for resolving complex biological structures. The average distance between adjacent fluorophores should be less than half the desired spatial resolution for optimal clarity. While this was not a major limitation in recent decades, the success of modern microscopy approaching molecular resolution down to the single-digit nanometer range will depend heavily on advancements in fluorescence labeling. This review highlights recent advances and challenges in labeling strategies for SRM, focusing on site-specific labeling technologies. These advancements are crucial for improving SRM precision and expanding our understanding of molecular interactions. [ABSTRACT FROM AUTHOR]

Abstract Metabolic labeling of proteins with non-canonical amino acids (ncAAs) provides unique bioorthogonal chemical groups during de novo synthesis by taking advantage of both endogenous and heterologous protein synthesis machineries. Labeled proteins can then be selectively conjugated to fluorophores, affinity reagents, peptides, polymers, nanoparticles or surfaces for a wide variety of downstream applications in proteomics and biotechnology. In this review, we focus on techniques in which proteins are residue- and site-specifically labeled with ncAAs containing bioorthogonal handles. These ncAA-labeled proteins are: readily enriched from cells and tissues for identification via mass spectrometry-based proteomic analysis; selectively purified for downstream biotechnology applications; or labeled with fluorophores for in situ analysis. To facilitate the wider use of these techniques, we provide decision trees to help guide the design of future experiments. It is expected that the use of ncAA labeling will continue to expand into new application areas where spatial and temporal analysis of proteome dynamics and engineering new chemistries and new function into proteins are desired.

As a consequence of their small size, high stability and high affinity, single domain antibody fragments (sdAbs) are appealing targeting vectors for radiopharmaceutical development. With sdAbs binding to internalizing receptors like HER2, residualizing prosthetic agents can enhance tumor retention of radioiodine, which until now has been done with random labeling approaches. Herein we evaluate a site-specific strategy utilizing a radioiodinated, residualizing maleimido moiety and the anti-HER2 sdAb 5F7 bearing a GGC tail for conjugation. Maleimidoethyl 3-(guanidinomethyl)-5-iodobenzoate ([131I]MEGMB) and its N -succinimidyl ester analogue, iso-[125I]SGMIB, were labeled by halodestannylation and conjugated with 5F7GGC and 5F7, respectively. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label experiments directly compared iso -[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC with regard to internalization/residualization and affinity on HER2-expressing SKOV-3 ovarian carcinoma cells as well as biodistribution and metabolite distribution in athymic mice with subcutaneous SKOV-3 xenografts. [131I]MEGMIB-5F7GGC had an immunoreactivity of 81.3% and K d = 0.94 ± 0.27 nM. Internalization assays demonstrated high intracellular trapping for both conjugates, For example, at 1 h, intracellular retention was 50.30 ± 3.36% for [131I]MEGMIB-5F7GGC and 55.95 ± 3.27% for iso -[125I]SGMIB-5F7, while higher retention was seen for iso -[125I]SGMIB-5F7 at later time points. Peak tumor uptake was similar for both conjugates (8.35 ± 2.66%ID/g and 8.43 ± 2.84%ID/g for iso -[125I]SGMIB-5F7 and [131I]MEGMIB-5F7GGC at 1 h, respectively); however, more rapid normal tissue clearance was seen for [131I]MEGMIB-5F7GGC, with a 2-fold higher tumor-to-kidney ratio and a 3-fold higher tumor-to-liver ratio compared with co-injected iso -[125I]SGMIB-5F7. Consisted with this, generation of labeled catabolites in the kidneys was higher for [131I]MEGMIB-5F7GGC. [131I]MEGMIB-5F7GGC offers similar tumor targeting as iso -[125I]SGMIB-5F7 but with generally lower normal tissue uptake. The site specific nature of the [131I]MEGMIB reagent may facilitate clinical translation, particularly for sdAb with compromised affinity after random labeling. [ABSTRACT FROM AUTHOR]

Transcription and translation obey to the genetic code of four nucleobases and 21 amino acids evolved over billions of years. Both these processes have been engineered to facilitate the use of non‐natural building blocks in both nucleic acids and proteins, enabling researchers with a decent toolbox for structural and functional analyses. Here, we review the most common approaches for how labeling of both nucleic acids as well as proteins in a site‐selective fashion with either modifiable building blocks or spectroscopic probes can be facilitated by genetic code expansion. We emphasize methodological approaches and how these can be adapted for specific modifications, both during as well as after biomolecule synthesis. These modifications can facilitate, for example, a number of different spectroscopic analysis techniques and can under specific circumstances even be used in combination. [ABSTRACT FROM AUTHOR]

Making peptide bonds is tightly controlled by genetic code and machinery which includes cofactors, ATP, and RNAs. In this regard, the stand-alone and genetic-code-independent peptide ligases constitute a new family of renegade peptide-bond makers. A prime example is butelase-1, an Asn/Asp(Asx)-specific ligase that structurally belongs to the asparaginyl endopeptidase family. Butelase-1 specifically recognizes a C-terminal Asx-containing tripeptide motif, Asn/Asp-Xaa-Yaa (Xaa and Yaa are any amino acids), to form a site-specific Asn-Xaa peptide bond either intramolecularly as cyclic proteins or intermolecularly as modified proteins. Our work in the past five years has validated that butelase-1 is a potent and versatile ligase. Here we review the advances in ligases, with a focus on butelase-1, and their applications in engineering bioactive peptides and precision protein modifications, antibody-drug conjugates, and live-cell labeling. [ABSTRACT FROM AUTHOR]

Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A‐T and G‐C base‐pairs are therefore uniquely suitable to facilitate position‐specific labeling of nucleic acids. Here, we employ the orthogonal kappa‐xanthosine‐base‐pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy‐version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7‐deaza‐xanthosine, which allows to site‐specifically modify transcribed RNA molecules with fluorophores or other labels. [ABSTRACT FROM AUTHOR]

Photosynthesis is one of the most fundamental and complex mechanisms in nature. It is a well‐studied process, however, some photosynthetic mechanisms are yet to be deciphered. One of the many proteins that take part in photosynthesis, cytochrome bd, is a terminal oxidase protein that plays a role both in photosynthesis and in respiration in various organisms, specifically, in cyanobacteria. To clarify the role of cytochrome bd in cyanobacteria, a system for the incorporation of an unnatural amino acid into a genomic membrane protein cytochrome bd was constructed in Synechococcus sp. PCC7942. N‐propargyl‐ l‐lysine (PrK) was incorporated into mutants of cytochrome bd. Incorporation was verified and the functionality of the mutant cytochrome bd was tested, revealing that both electrochemical and biochemical activities were relatively similar to those of the wild‐type protein. The incorporation of PrK was followed by a highly specific labeling and localization of the protein. PrK that was incorporated into the protein enabled a "click" reaction in a bio‐orthogonal manner through its alkyne group in a highly specific manner. Cytochrome bd was found to be localized mostly in thylakoid membranes, as was confirmed by an enzyme‐linked immunosorbent assay, indicating that our developed localization method is reliable and can be further used to label endogenous proteins in cyanobacteria. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus’s conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of IAV.

Synthetic near-infrared-II (NIR-II) dyes are promising for deep tissue imaging, yet they are generally difficult to target a given biomolecule with high specificity. Furthermore, the interaction mechanism between albumin and cyanine molecules, which is usually regarded as uncertain "complexes" such as crosslinked nanoparticles, remains poorly understood. Methods: Here, we propose a new class of NIR-II fluorogenic dyes capable of site-specific albumin tagging for in situ albumin seeking/targeting or constructing high-performance cyanine@albumin probes. We further investigate the interaction mechanism between NIR-II fluorogenic dyes and albumin. Results: We identify CO-1080 as an optimal dye structure that produces a stable/bright NIR-II cyanine@albumin probe. CO-1080 exhibits maximum supramolecular binding affinity to albumin while catalyzing their covalent attachment. The probe shows exact binding sites located on Cys476 and Cys101, as identified by proteomic analysis and docking modeling. Conclusion: Our cyanine@albumin probe substantially improves the pharmacokinetics of its free dye counterpart, enabling high-performance NIR-II angiography and lymphography. Importantly, the site-specific labeling tags between NIR-II fluorogenic dyes and albumin occur under mild conditions, offering a specific and straightforward synthesis strategy for NIR-II fluorophores in the fields of targeting bioimaging and imaging-guided surgery., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Most single-molecule techniques observing RNA in vitro or in vivo require fluorescent labels that have to be connected to the RNA of interest. In recent years, a plethora of methods has been developed to achieve site-specific labelling, in many cases under near-native conditions. Here, we review chemical as well as enzymatic labelling methods that are compatible with single-molecule fluorescence spectroscopy or microscopy and show how these can be combined to offer a large variety of options to site-specifically place one or more labels in an RNA of interest. By either chemically forming a covalent bond or non-covalent hybridization, these techniques are prerequisites to perform state-of-the-art single-molecule experiments. [ABSTRACT FROM AUTHOR]

Purpose: Construction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system.Procedures: We used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively.Results: Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts.Conclusions: These results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities. [ABSTRACT FROM AUTHOR]

Chemical synthesis is a well‐established method for the preparation in the research laboratory of multiple‐tens‐of‐milligram amounts of correctly folded, high purity protein molecules. Chemically synthesized proteins enable a broad spectrum of novel protein science. Racemic mixtures consisting of d‐protein and l‐protein enantiomers facilitate crystallization and determination of protein structures by X‐ray diffraction. d‐Proteins enable the systematic development of unnatural mirror image protein molecules that bind with high affinity to natural protein targets. The d‐protein form of a therapeutic target can also be used to screen natural product libraries to identify novel small molecule leads for drug development. Proteins with novel polypeptide chain topologies including branched, circular, linear‐loop, and interpenetrating polypeptide chains can be constructed by chemical synthesis. Medicinal chemistry can be applied to optimize the properties of therapeutic protein molecules. Chemical synthesis has been used to redesign glycoproteins and for the a priori design and construction of covalently constrained novel protein scaffolds not found in nature. Versatile and precise labeling of protein molecules by chemical synthesis facilitates effective application of advanced physical methods including multidimensional nuclear magnetic resonance and time‐resolved FTIR for the elucidation of protein structure–activity relationships. The chemistries used for total synthesis of proteins have been adapted to making artificial molecular devices and protein‐inspired nanomolecular constructs. Research to develop mirror image life in the laboratory is in its very earliest stages, based on the total chemical synthesis of d‐protein forms of polymerase enzymes. [ABSTRACT FROM AUTHOR]

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with 18F-FBEM. 18F-FBEM was synthesized by coupling 18F-fluorobenzoic acid (18F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of 18F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting 18F-FBEM, non-decay corrected). The radiochemical purity of 18F-FBEM-Cys-Annexin V was >95%. The specific radioactivities of 18F-FBEM and 18F-FBEM-Cys-Annexin V were >150 and 3.17 GBq/µmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel 18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of 18F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent.

Abstract: Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging. [ABSTRACT FROM AUTHOR]

Atypical inteins have been used for protein site-specific labeling and modification. S1 or S11 intein contain a small N-intein or C-intein which can be chemically synthesized and added with desired labels or modifications. However, seldom reports have been found to develop multi-protein specific labeling in one system at the same time. Here, we established a specific labeling method for two proteins based on three pairs of atypical inteins, RBS1 ( Rma DnaB S1)/SGS1 ( Ssp GyrB S1) intein, TE3S11 ( Ter DnaE-3 S11)/SGS11 ( Ssp GyrB S11) intein, and RBS1/TE3S11 intein. Firstly, intein fragments were respectively fused with model proteins, expressed and purified as precursors. The non-cross reactivity between inteins was then determined by splicing reactions analysis through Western-blot. Finally, the model proteins were specifically labeled with fluorescent groups in one system mediating these intein pairs, and can be labeled even in the cell lysate. Our results for the first time report a method labeling the N/C-terminal of two proteins altogether in the same system based on above intein pairs with a marked splicing efficiency, which could potentially be used for protein structural and functional research or some specific applications. [ABSTRACT FROM AUTHOR]

Protein biogenesis is poorly understood due to the ribosome that perturbs measurement attempted on the ribosome-bound nascent chain (RNC). Investigating nascent chain dynamics may provide invaluable insight into the co-translational processes such as structure formation or interaction with a chaperone [e.g., the bacterial trigger factor (TF)]. In this study, we aim to establish a platform for studying nascent chain dynamics by exploring the local environment near the fluorescent dye on site-specifically labeled RNCs with time-resolved fluorescence anisotropy. To prepare a quantitative model of fluorescence depolarization, we utilized intrinsically disordered protein bound to ribosome, which helped us couple the sub-nanosecond depolarization with the motion of the nascent chain backbone. This was consistent with zinc-finger-domain-containing RNCs, where the extent of sub-nanosecond motion decreased upon the addition of zinc when the fluorophore was in close proximity of the domain. After the characterization of disordered nascent chain dynamics, we investigated the synthesis of a model cytosolic protein, Entner–Doudoroff aldolase, labeled at different sites during various stages of translation. Depending on the stage of translation, the addition of the TF to the nascent chain led to two different responses in the nascent chain dynamics serendipitously, suggesting steric hindrance between the nascent chain and the chaperone as a mechanism for TF dissociation from the ribosome during translation. Overall, our study demonstrates the possible use of site-specific labeling and time-resolved anisotropy to gain insight on chaperone binding event at various stages of translation and hints on TF co-translational mechanism. [ABSTRACT FROM AUTHOR]

Novel difunctional initiators that incorporate Förster/fluorescence resonance energy transfer (FRET) pairs are generated to carry out atom transfer radical polymerization of styrene, methyl methacrylate, and n-butyl methacrylate monomers by an efficient manner. Based on the chemical structures of the initiators, the locations of the fluorophore moiety are dictated to be in the center of the chain with accurately quantified chain functionality (>90% labeling ratio). The site-specific integration of FRET dyes into separate polymer chain centers allows for characterization of the well-defined interchain distance quantitatively based on the response between these fluorescent probes. The reliability of this technique is verified in bulk state, which is in well agreement with the theoretical ones. This well-defined FRET system is expected to be a promising candidate to provide a distinct physical image at a microscopic level regarding scaling chain dimension, chain interpenetration, and polymer compatibility. [ABSTRACT FROM AUTHOR]

The transcription factor NF-ĸB is a central mediator of immune and inflammatory responses. To understand the regulation of NF-ĸB, it is important to probe the underlying thermodynamics, kinetics, and conformational dynamics of the NF-ĸB/IĸBα/DNA interaction network. The development of genetic incorporation of non-canonical amino acids (ncAA) has enabled the installation of biophysical probes into proteins with site specificity. Recent single-molecule FRET (smFRET) studies of NF-ĸB with site-specific labeling via ncAA incorporation revealed the conformational dynamics for kinetic control of DNA-binding mediated by IĸBα. Here we report the design and protocols for incorporating the ncAA p-azidophenylalanine (pAzF) into NF-ĸB and site-specific fluorophore labeling with copper-free click chemistry for smFRET. We also expanded the ncAA toolbox of NF-ĸB to include p-benzoylphenylalanine (pBpa) for UV crosslinking mass spectrometry (XL-MS) and incorporated both pAzF and pBpa into the full-length NF-ĸB RelA subunit which includes the intrinsically disordered transactivation domain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This review focuses on the various approaches to covalently attach a chromophore to a biomolecule of interest in site-specific manner. Novel methods like inverse electron-demand Diels-Alder reaction, Pictet-Spengler ligation and enzyme tags like SNAP and Halo-tags are critically discussed and compared to established techniques like copper-free click reaction and native chemical ligation. Selected examples in which the tags have been exploited for in vitro or in vivo imaging are reviewed and evaluated. [ABSTRACT FROM AUTHOR]

Placement of a tag sequence is usually limited to either terminal end of the target protein, reducing the potential of epitope tags for various labeling applications. The PA tag is a dodecapeptide (GVAMPGAEDDVV) that is recognized by a high-affinity antibody NZ-1. We determined the crystal structure of the PA-tag-NZ-1 complex and found that NZ-1 recognizes a central segment of the PA tag peptide in a tight β-turn configuration, suggesting that it is compatible with the insertion into a loop. This possibility was tested and confirmed using multiple integrin subunits and semaphorin. More specifically, the PA tag can be inserted at multiple locations within the integrin αIIb subunit (encoded by ITGA2B) of the fibrinogen receptor αIIbβ3 integrin (of which the β3 subunit is encoded by ITGB3) without affecting the structural and functional integrity, while maintaining its high affinity for NZ-1. The large choice of the sites for 'epitope grafting' enabled the placement of the PA tag at a location whose accessibility is modulated during the biological action of the receptor. Thus, we succeeded in converting a general anti-tag antibody into a special anti-integrin antibody that can be classified as a ligand-induced binding site antibody. [ABSTRACT FROM AUTHOR]

Spatial proteomics has recently garnered significant interest, as it offers to provide unprecedented insight into biological processes in both health and disease, by connecting protein expression patterns from the subcellular level to the tissue or even organism level. These high-content approaches generally rely on a high degree of multiplexing, whereby multiple proteins can be detected simultaneously. The most versatile multiplexing approaches utilize antibodies to confer specificity for various intracellular proteins of interest. Therefore, these methods must be able to differentiate many antibodies at once. In this chapter, we describe a simple and rapid approach to labeling antibodies with distinct epitope tags in a site-specific manner. This allows multiple antibodies, even from the same host species, to be uniquely identified and detected and offers a simple approach for spatial proteomic applications., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We present in-membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G-protein-coupled receptors (GPCRs) with minimal mutagenesis. This method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site-specific introduction of chromophores for NMR and fluorescence spectroscopy in the human κ-opioid receptor (KOR) and the human A2A adenosine receptor (A2AAR). IMCM is applicable to a wide range of invitro studies of GPCRs, including single-molecule spectroscopy, and is a promising platform for in-cell spectroscopy experiments. [ABSTRACT FROM AUTHOR]

Split-inteins are a subset of inteins that are expressed in two separate halves and catalyze splicing in trans upon association of the two domains. They occur naturally and have also been artificially generated by splitting of contiguous ones. With their unique properties, split-inteins offer improved controllability, flexibility and capability to existing tools based on contiguous inteins. In addition, split-inteins have proven useful in several new applications. This review gives a general introduction to split-inteins with a focus on their role in expanding the applications of intein-based technologies. [ABSTRACT FROM AUTHOR]

Site-specific labeling of synthetic peptides carrying N-methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N-terminus was synthesized by the solid-phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N-terminus, leaving side chain amino group intact. The synthetic human β-defensin-2 carrying MeOGly at its N-terminus or the side chain amino group of Lys10 reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N-methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site-specific labeling of linear synthetic peptides as well as disulfide-containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A novel annexin V derivative (Cys-Annexin V) with a single cysteine residue at its C-terminal has been developed and successfully labeled site-specifically with 18F-FBEM. 18F-FBEM was synthesized by coupling 18F-fluorobenzoic acid (18F-FBA) with N-(2-aminoethyl)maleimide using optimized reaction conditions. The yield of 18F-FBEM-Cys-Annexin V was 71.5% ± 2.0% (n = 4, based on the starting 18F-FBEM, non-decay corrected). The radiochemical purity of 18F-FBEM-Cys-Annexin V was >95%. The specific radioactivities of 18F-FBEM and 18F-FBEM-Cys-Annexin V were >150 and 3.17 GBq/μmol, respectively. Like the 1st generation 18F-SFB-Annexin V, the novel 18F-FBEM-Cys-Annexin V mainly shows renal and to a lesser extent, hepatobiliary excretion in normal mice. In rat hepatic apoptosis models a 3.88 ± 0.05 (n = 4, 1 h) and 10.35 ± 0.08 (n = 4, 2 h) increase in hepatic uptake of 18F-FBEM-Cys-Annexin V compared to normal rats was observed after injection via the tail vein. The liver uptake ratio (treated/control) at 2 h p.i. as measured via microPET correlated with the ratio of apoptotic nuclei in liver observed using TUNEL histochemistry, indicating that the novel 18F-FBEM-Cys-Annexin V is a potential apoptosis imaging agent. [ABSTRACT FROM AUTHOR]

Site-specific 13C isotope labeling is a useful approach that allows for the measurement of homonuclear 13C,13C coupling constants. For three site-specifically labeled oligosaccharides, it is demonstrated that using the J-HMBC experiment for measuring heteronuclear long-range coupling constants is problematical for the carbons adjacent to the spin label. By incorporating either a selective inversion pulse or a constant-time element in the pulse sequence, the interference from one-bond 13C,13C scalar couplings is suppressed, allowing the coupling constants of interest to be measured without complications. Experimental spectra are compared with spectra of a nonlabeled compound as well as with simulated spectra. The work extends the use of the J-HMBC experiments to site-specifically labeled molecules, thereby increasing the number of coupling constants that can be obtained from a single preparation of a molecule. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

NMR spectroscopy is the leading technique for determining glycans' three-dimensional structure and dynamic in solution as well as a fundamental tool to study protein-glycan interactions. To overcome the severe chemical shift degeneracy of these compounds, synthetic probes carrying NMR-active nuclei (e. g., 13 C or 19 F) or lanthanide tags have been proposed. These elegant strategies permitted to simplify the complex NMR analysis of unlabeled analogues, shining light on glycans' conformational aspects and interaction with proteins. Here, we highlight some key achievements in the synthesis of specifically labeled glycan probes and their contribution towards the fundamental understanding of glycans., (© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Thiamine pyrophosphate (TPP)-sensitive mRNA domains are the most prevalent riboswitches known. Despite intensive investigation, the complex ligand recognition and concomitant folding processes in the TPP riboswitch that culminate in the regulation of gene expression remain elusive. Here, we used single-molecule fluorescence resonance energy transfer imaging to probe the folding landscape of the TPP aptamer domain in the absence and presence of magnesium and TPP. To do so, distinct labeling patterns were used to sense the dynamics of the switch helix (P1) and the two sensor arms (P2/P3 and P4/P5) of the aptamer domain. The latter structural elements make interdomain tertiary contacts (L5/P3) that span a region immediately adjacent to the ligand-binding site. In each instance, conformational dynamics of the TPP riboswitch were influenced by ligand binding. The P1 switch helix, formed by the 5' and 3' ends of the aptamer domain, adopts a pre- dominantly folded structure in the presence of Mg2+ alone. However, even at saturating concentrations of Mg2+ and TPP, the P1 helix, as well as distal regions surrounding the TPP-binding site, exhibit an unexpected degree of residual dynamics and disperse kinetic behaviors. Such plasticity results in a persistent exchange of the P3/PS forearms between open and closed configurations that is likely to facilitate entry and exit of the TPP ligand. Correspondingly, we posit that such features of the TPP aptamer domain contribute directly to the mechanism of riboswitch-mediated translational regulation. [ABSTRACT FROM AUTHOR]

Hepcidin is a small disulfide-rich peptide hormone that plays a key role in the regulation of iron homeostasis by binding and mediating the degradation of the cell membrane iron efflux transporter, ferroportin. Since it is a small peptide, chemical synthesis is a suitable approach for the preparation of mature human hepcidin. However, oxidative folding of synthetic hepcidin is extremely difficult due to its high cysteine content and high aggregation propensity. To improve its oxidative folding efficiency, we propose a reversible S-modification approach. Introduction of eight negatively charged sulfonate moieties into synthetic hepcidin significantly decreased its aggregation propensity and, under optimized conditions, dramatically increased the refolding yield. The folded hepcidin displayed a typical disulfide-constrained β-sheet structure and could induce internalization of enhanced green fluorescent protein (EGFP) tagged ferroportin in transfected HEK293 cells. In order to study interactions between hepcidin and its receptor ferroportin, we propose a general approach for site-specific labeling of synthetic hepcidin analogues by incorporation of an l-propargylglycine during chemical synthesis. Following efficient oxidative refolding, a hepcidin analogue with Met20 replaced by l-propargylglycine was efficiently mono-labeled by a red fluorescent dye through click chemistry. The labeled hepcidin was internalized into the transfected cells together with the EGFP-tagged ferroportin, suggesting direct binding between hepcidin and ferroportin. The labeled hepcidin was also a suitable tool to visualize internalization of overexpressed or even endogenously expressed ferroportin without tags. We anticipate that the present refolding and labeling approaches could also be used for other synthetic peptides. [ABSTRACT FROM AUTHOR]

Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6×His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies. [ABSTRACT FROM AUTHOR]