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New Biarsenical Ligands and Tetracysteine Motifs for Protein Labeling in Vitro and in Vivo: Synthesis and Biological Applications
Abstract
We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.
... In this regard, it would be particularly valuable to develop a way to monitor GAPDH heme binding and transfer events. To this end, we designed a reporter construct called tetra-Cys human GAPDH (TC-hGAPDH) which, following its expression and labeling with a commercially-available FlAsH reagent [22], could indicate GAPDH heme binding by fluorescence quenching in either its purified form or when it is expressed in mammalian cells. Here, we characterize TC-hGAPDH and demonstrate its utility as a probe for visualizing GAPDH heme binding in living cells and for studying GAPDH-dependent heme transfers, in this case to the client protein IDO1, a heme-containing redox enzyme whose activity influences many biological processes including inflammation and immune surveillance of cancer growth [23][24][25]. ...
... To enable monitoring of GAPDH heme binding we adopted FlAsHbased technology [22] like we successfully utilized before to study heme binding by the hemeprotein sGCβ [19]. This involved inserting a short DNA sequence into our human GAPDH expression plasmids that encode for a FlAsH bi-arsenical-binding tetra-cysteine (TC) motif immediately following His57 (Fig. 1A). ...
... This location is in a loop region downstream from the presumed heme-coordinating His53 in GAPDH [30], thus creating TC-hGAPDH (sequence details in Fig. S1). The TC motif is designed to bind the bi-arsenical dye FlAsH (Fig. 1B), which only fluoresces after it becomes bound to the TC sequence in a protein [22,34]. The TC-hGAPDH construct we designed for bacterial expression also contained a C-terminal GST fusion protein to aid in purification [30]. ...
Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45–65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.
... 9,10 Alternatively, Tsien and coworkers developed small organic fluorophores, termed FlAsH and ReAsH, that are activated by binding to specific tetracysteine motifs (e.g., CCPGCC). [11][12][13] This method allows the visualization of intracellular trafficking of recombinant proteins without the washing step. 14,15 However, these arsenic probes have an ambiguously toxic impact on living cells, 16,17 along with the uncertainty resulting from reversibility, 13,16,18 limiting their applications. ...
... [11][12][13] This method allows the visualization of intracellular trafficking of recombinant proteins without the washing step. 14,15 However, these arsenic probes have an ambiguously toxic impact on living cells, 16,17 along with the uncertainty resulting from reversibility, 13,16,18 limiting their applications. Nevertheless, the small molecule protein-labeling strategy with a non-toxic fluorogenic agent would be promising to monitor enzymatic productivity. ...
... Chromatography on silica gel column using dichloromethane as the eluent afforded product as white solid. 1 Figure S9). 13 Figure S11). Figure S12). ...
A rapid and convenient strategy to monitor the productivity of biomanufacturing is essential for the research in optimizing relevant bioprocesses. In this work, we have developed a fluorescein‐derived probe (FL‐DT) that reacts rapidly with thiol groups via 1,4‐Michael addition reaction of the sulfhydryl to unsaturated ketone and releases fluorescence. FL‐DT specifically forms fluorescent adduct with two adjacent thiols in a protein of interest (POI), making the probe a reliable tool for protein quantification. The production of xylanase fused with a short di‐Cys tag was then successfully monitored and quantified with FL‐DT in Escherichia coli system under different protein expression conditions, providing useful information for optimizing the bioprocess. Our work provides a convenient and efficient strategy for POI labeling and monitoring bioproduction.
... The amplified PCR products were mixed with the linearized pRS424-FLDV2 by XhoI (at 5427-5430 nt position) or pRS424-FLDV1 by NheI (at 5877-5890 nt position) to form Saccharomyces cerevisiae YPH857 competent cells. The homologous recombination-occurred plasmid-contained S. cerevisiae was selected and amplified in Tryptophan-lacked yeast media, followed by transforming and expanding E. coli stbl2 competent cells in ampicillin-contained LB media (30,31). The designed homologous recombination as well as the incomplete replacements happened by chance in yeast cells. ...
... Infectious NS3-chimeric DENV cDNAs were linearized by SacII (for DENV1 backbone), or BcgI or SacI (for DENV2 backbone) at the 3' ends. The linearized cDNAs were used as templates for in vitro transcription reaction to synthesize 5' cap infectious RNAs with SP6 RNA polymerase and m 7 GpppG cap analog (30,31). Each synthesized RNA (~3 µg) was electroporated into ~1 x 10 6 BHK-21 cells (American Type Culture Collection), using Amaxa Nucleofector II system (Amaxa). ...
Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%–78% amino acid identities, resulting in incomplete immunity, where one serotype’s infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5’ RNA triphosphatase (5’RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations’ frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells.
IMPORTANCE
Enzyme activities for replicating DENV 5’ cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5’ cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)–NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.
... Griffin et al. pioneered the use of biarsenical dyes for the in vivo labelling of target peptides or proteins with tiny fluorescent dyes (Scheme 16) [90]. A fluorescent dye containing two arsenic moieties binds to a genetically encoded pattern of four cysteines in the sequence Cys-Cys-Xaa-Xaa-Cys-Cys with high affinity in this approach (where Xaa represents any amino acid other than cysteine) [91]. Two As(III) substituents in the cell permeable fluorescent dye FlAsH [4′,5′-bis(1,3,2,dithioarsolan-2-yl)fluorescein] (45) pair with the four cysteine thiol groups in the motif. ...
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... We also included DNA sequences encoding a tetracysteine tag (-CCPGCC-) upstream of the enterokinase site. The tetracysteine peptide tag could bind to the biarsenical ligand ReAsH to give a red fluorescent signal (16), providing a readout for real-time peptide synthesis in IVTT. In addition, we designed a biotinylated forward primer and a fluorescently labeled reverse primer (Table S2) for amplification of the DNA template in preparation for attaching fluorescent barcodes to the APCs. ...
Screening for peptide fragments that can be displayed on antigen-presenting cells is an essential step in vaccine development. The current approach for this process is slow and costly as it involves separately pulsing cells with chemically synthesized peptides. We present Microfluidic-Enabled production of DNA-barcoded APC Library (MEDAL), a high throughput microfluidic droplet platform for parallel production of DNA-barcoded APCs loaded with enzymatically synthesized peptides. Droplets containing peptides and their encoding DNA are produced from microfluidic PCR-IVTT reaction. APCs presenting both peptides and DNA barcodes are obtained by injecting cells into these droplets. Up to 9,000 different APCs can be produced and screened within a 10-hour workflow. This approach allowed us to identify peptide sequences that bind to APCs expressing H-2Kb MHC class 1 molecule with next-generation sequencing of DNA barcodes.
... the outer mitochondrial membrane that has previously been implicated in mitochondrial heme export in erythrocytes during erythropoiesis 20 . We performed experiments in a human embryonic kidney cell line (HEK293T cells) transfected to express an HA-tagged human GAPDH reporter protein called TC-hGAPDH 19 , which after being labeled with FlAsH reagent 19,21 signals its heme binding in living cells or in solution by a fluorescence quenching of its FlAsH signal 19 . We determined how siRNA-targeted knockdown of cell FLVCR1b expression impacts heme loading onto TC-GAPDH in living cells in response to our stimulating their mitochondrial heme biosynthesis by providing the heme precursor molecules δ-aminolevulinic acid (δ-ALA) and ferric citrate (Fe) 22 , which we have previously shown causes the total heme level in the HEK293T cells, and the level of heme bound in the TC-hGAPDH, to increase by about 3-fold within 2 h 19 . ...
Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.
... The core structure of the tetra-cysteine motif is Cys-Cys-Xaa-Xaa-Cys-Cys (CCXXCC), where "X" represents any amino acid, although a preference is often given to Pro-Gly (Adams et al., 2002;Martin et al., 2005). In this arrangement, the peptide forms a hairpin structure that positions the cysteine residues in a manner conducive to interacting with the arsenic atoms present in the fluorescent probe. ...
Host-pathogen interactions play a critical role in infectious diseases, and understanding the underlying mechanisms is vital for developing effective therapeutic strategies. The visualization and characterization of bacterial proteins within host cells is key to unraveling the dynamics of these interactions. Various protein labeling strategies have emerged as powerful tools for studying host-pathogen interactions, enabling the tracking, localization, and functional analysis of bacterial proteins in real-time. However, the labeling and localization of Salmonella secreted type III secretion system (T3SS) effectors in host cells poses technical challenges. Conventional methods disrupt effector stoichiometry and often result in non-specific staining. Bulky fluorescent protein fusions interfere with effector secretion, while other tagging systems such as 4Cys-FLaSH/Split-GFP suffer from low labeling specificity and a poor signal-to-noise ratio. Recent advances in state-of-the-art techniques have augmented the existing toolkit for monitoring the translocation and dynamics of bacterial effectors. This comprehensive review delves into the bacterial protein labeling strategies and their application in imaging host-pathogen interactions. Lastly, we explore the obstacles faced and potential pathways forward in the realm of protein labeling strategies for visualizing interactions between hosts and pathogens.
... Fluorescence-based techniques have greatly facilitated the study of protein localization, structure and function in living systems 5 . Genetic fusion has been widely used for labeling POIs, including but not limited to the fusion of fluorescent proteins (FPs), diverse self-labeling enzymes 6-10 and peptide tags for subsequent labeling [11][12][13] . However, these strategies generally require overexpression of the open reading frame (ORF) encoding the POI and are typically limited to protein fusion at C-or N-termini and flexible loops, which may perturb the expression, folding, localization and function of the POI 14 . ...
Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.
... To overcome these drawbacks, we have developed a highly specific and minimally disruptive labeling strategy for studying antigen-specific T cell-APC interactions. Our approach involves introducing a six-amino acid tetracysteine tag (CCPGCC) on antigenic peptides and inducing fluorescence using membrane permeable thiol-reactive Arsenical Hairpin (AsH) probes (Hoffmann et al., 2010;Adams et al., 2002). We have engineered the OVA (323)(324)(325)(326)(327)(328)(329)(330)(331)(332)(333)(334)(335)(336)(337)(338)(339) peptide by placing the tetracysteine tag at either the amino or the carboxy terminus, away from the TCR and MHC binding sites, to avoid disrupting T cell-APC synapse in vitro and in vivo (Robertson et al., 2000). ...
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
... FlAsH is a membrane-permeable fluorogenic molecule that binds specifically with the TC tag (Cys-Cys-Pro-Gly-Cys-Cys) and which turns on fluorescence upon binding. [12][13][14][15] We sought to introduce this tag into the major GV structural protein, GvpA, such that expressed intracellular GVs would be able to bind FlAsH and turn on fluorescence. We screened for TC-containing GvpA mutants in bacteria and, once we identified a suitable variant, expressed TC-tagged Anabaena flosaquae GVs ("AnaGVs") in HEK 293T cells. ...
Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' sub-cellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon binding the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag, and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. We used fluorescence images of the tagged GVs to study GV size and distance distributions within these cells. This bioorthogonal labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.
... It may be determined directly from the amino acid type [41]. This property is critical when designing sequences optimized for high synthesis yield and purity [42]. The training was performed for each complex using the acid dissociation constants from the receptor and the first half of the ligand's dissociation constants. ...
This paper presents a new approach for protein generation based on one-shot learning and hybrid quantum neural networks. Given a single protein complex, the system learns how to predict the remaining unknown properties, without resorting to autoregression, from the physicochemical properties of the receptor and a prior on the physicochemical properties of the ligand. In contrast with other approaches, QuantumBound learns from a single instance, not from a large dataset, as is common in deep learning. By knowing half of the properties of the ligand, the system can predict the remaining half with an average relative error of 1.43% for a dataset consisting of one hundred and twenty Covid-19 spikes complexes. To the best of our knowledge, this is the first time that one-shot learning and hybrid quantum computing have been applied to protein generation.
... To overcome these drawbacks, we have developed a highly speci c and minimally disruptive labeling strategy for studying antigen-speci c T cell-APC interactions. Our approach involves introducing a sixamino acid tetracysteine tag (CCPGCC) on antigenic peptides and inducing uorescence using membrane permeable thiol-reactive Arsenical Hairpin (AsH) probes [5,8]. We have engineered the OVA (323-339) peptide by placing the tetracysteine tag at either the amino or the carboxy terminus, away from the TCR and MHC binding sites, to avoid disrupting T cell-APC synapse in vitro and in vivo [9]. ...
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
... The fluorescein derivative FlAsH-EDT 2 with two As(III) substituents can specifically react with the tetracysteine (TC) tag (FLNCCPGCCMEP) (Adams et al., 2002;Martin et al., 2005) that is fused to the C-terminus of a target protein ( Supplementary Fig. S5A). FlAsH-EDT 2 cannot penetrate the bacterial outer membrane, and it has been utilized to screen high-producing strains on microtiter plates (Haitjema et al., 2014). ...
... To gain a better understanding of where functional Arf1 localizes, we utilized the FlAsH tag (Griffin et al., 1998). FlAsH is a fluorogenic, small molecule that binds short tetracysteine (TetCys) motifs that consist of CCXXCC, where XX represents any two amino acids but is ideally PG (Adams et al., 2002). Importantly, FlAsH binding to the TetCys motifs causes a substantial increase in green fluorescence, allowing visualization of tagged proteins within their cellular context. ...
Arf GTPases are central regulators of the Golgi complex, which serves as the nexus of membrane trafficking pathways in eukaryotic cells. Arf proteins recruit dozens of effectors to modify membranes, sort cargos, and create and tether transport vesicles, and are therefore essential for orchestrating Golgi trafficking. The regulation of Arf activity is controlled by the action of Arf-GEFs, which activate via nucleotide exchange, and Arf-GAPs, which inactivate via nucleotide hydrolysis. The localization dynamics of Arf GTPases and their Arf-GAPs during Golgi maturation have not been reported. Here we use the budding yeast model to examine the temporal localization of the Golgi Arf-GAPs. We also determine the mechanisms used by the Arf-GAP Age2 to localize to the Golgi. We find that the catalytic activity of Age2 and a conserved sequence in the unstructured C-terminal domain of Age2 are both required for Golgi localization. This sequence is predicted to form an amphipathic helix and mediates direct binding of Age2 to membranes in vitro. We also report the development of a probe for sensing active Arf1 in living cells and use this probe to characterize the temporal dynamics of Arf1 during Golgi maturation.
... As the protein part can be targeted by using specific promoters or genetic fusion to organelle marker proteins, researchers can control the localization of organic molecules by simply adding the compound with a corresponding ligand moiety to the tissue under investigation. Examples of self-labeling tag proteins include HaloTag [51], SNAP-tag [52], PYP-tag [53], eDHFR [54], FlAsH and ReAsH systems [55,56], FlARe [57], and FAST [58] tags. Among those systems, SNAP-tag and HaloTag proteins are the prototypical examples of systems in which a covalent bond is formed to a small molecule, while FAST system is a prototypical example of system in which a small molecule non-covalently binds to protein. ...
Fluorescent protein (FP)-based biosensors are genetically encoded tools that enable the imaging of biological processes in the context of cells, tissues, or live animals. Though widely used in biological research, practically all existing biosensors are far from ideal in terms of their performance, properties, and applicability for multiplexed imaging. These limitations have inspired researchers to explore an increasing number of innovative and creative ways to improve and maximize biosensor performance. Such strategies include new molecular biology methods to develop promising biosensor prototypes, high throughput microfluidics-based directed evolution screening strategies, and improved ways to perform multiplexed imaging. Yet another approach is to effectively replace components of biosensors with self-labeling proteins, such as HaloTag, that enable the biocompatible incorporation of synthetic fluorophores or other ligands in cells or tissues. This mini-review will summarize and highlight recent innovations and strategies for enhancing the performance of FP-based biosensors for multiplexed imaging to advance the frontiers of research.
... The constructs were induced with 5 nM aTc at 16 hpi. At 18 hpi, tetracysteine tag was stained with ReAsH-EDT 2 by using the TC-ReAsH II in-cell tetracysteine tag detection kit (Thermo Scientific, Waltham, MA) (53,54). Briefly, infected cells were washed three times with 1Â HBSS with Ca 21 and Mg 21 (Gibco). ...
Chlamydia trachomatis is an obligate intracellular bacterial pathogen. In evolving to the intracellular niche, Chlamydia has reduced its genome size compared to other bacteria and, as a consequence, has a number of unique features. For example, Chlamydia engages the actin-like protein MreB, rather than the tubulin-like protein FtsZ, to direct peptidoglycan (PG) synthesis exclusively at the septum of cells undergoing polarized cell division. Interestingly, Chlamydia possesses another cytoskeletal element-a bactofilin ortholog, BacA. Recently, we reported BacA is a cell size-determining protein that forms dynamic membrane-associated ring structures in Chlamydia that have not been observed in other bacteria with bactofilins. Chlamydial BacA possesses a unique N-terminal domain, and we hypothesized this domain imparts the membrane-binding and ring-forming properties of BacA. We show that different truncations of the N terminus result in distinct phenotypes: removal of the first 50 amino acids (ΔN50) results in large ring structures at the membrane whereas removal of the first 81 amino acids (ΔN81) results in an inability to form filaments and rings and a loss of membrane association. Overexpression of the ΔN50 isoform altered cell size, similar to loss of BacA, suggesting that the dynamic properties of BacA are essential for the regulation of cell size. We further show that the region from amino acid 51 to 81 imparts membrane association as appending it to green fluorescent protein (GFP) resulted in the relocalization of GFP from the cytosol to the membrane. Overall, our findings suggest two important functions for the unique N-terminal domain of BacA and help explain its role as a cell size determinant. IMPORTANCE Bacteria use a variety of filament-forming cytoskeletal proteins to regulate and control various aspects of their physiology. For example, the tubulin-like FtsZ recruits division proteins to the septum whereas the actin-like MreB recruits peptidoglycan (PG) synthases to generate the cell wall in rod-shaped bacteria. Recently, a third class of cytoskeletal protein has been identified in bacteria-bactofilins. These proteins have been primarily linked to spatially localized PG synthesis. Interestingly, Chlamydia, an obligate intracellular bacterium, does not have PG in its cell wall and yet possesses a bactofilin ortholog. In this study, we characterize a unique N-terminal domain of chlamydial bactofilin and show that this domain controls two important functions that affect cell size: its ring-forming and membrane-associating properties.
... Cysteine is an essential amino acid that plays a critical role in numerous biochemical processes, including oxidative folding, peptide structure stabilization, catalysis, metal binding, and cell signaling [10][11][12][13][14][15]. Disulfide bonds, formed between two cysteine residues, are crucial for the three-dimensional structure, biological activity, chemical composition, biophysical properties, and proteolytic stability of peptides and proteins [16][17][18][19][20][21][22][23]. ...
In the chemical synthesis of conotoxins with multiple disulfide bonds, the oxidative folding process can result in diverse disulfide bond connectivities, which presents a challenge for determining the natural disulfide bond connectivities and leads to significant structural differences in the synthesized toxins. Here, we focus on KIIIA, a μ-conotoxin that has high potency in inhibiting Nav1.2 and Nav1.4. The non-natural connectivity pattern (C1—C9, C2—C15, C4—C16) of KIIIA exhibits the highest activity. In this study, we report an optimized Fmoc solid-phase synthesis of KIIIA using various strategies. Our results indicate that free random oxidation is the simplest method for peptides containing triple disulfide bonds, resulting in high yields and a simplified process. Alternatively, the semi-selective strategy utilizing Trt/Acm groups can also produce the ideal isomer, albeit with a lower yield. Furthermore, we performed distributed oxidation using three different protecting groups, optimizing their positions and cleavage order. Our results showed that prioritizing the cleavage of the Mob group over Acm may result in disulfide bond scrambling and the formation of new isomers. We also tested the activity of synthesized isomers on Nav1.4. These findings provide valuable guidance for the synthesis of multi-disulfide-bonded peptides in future studies.
... To overcome this problem, we used the FlAsH fluorophore, 4',5'-bis(1,3,2 dithioarsolan-2-yl) fluorescein to sitedirect label the SBDS protein. This is an arsenoxide dye with a high affinity for four spaced cysteines in a motif know as FlAsH-tag consisting of the sequence CCXXCC where X is any amino acid other than cysteine 16,17 . This tetracysteine motif is added to the N-or C-terminus of the protein by genetic engineering together with an appropriate linker to prevent the disruption of the overall fold of the protein. ...
... We expressed the DNA restriction enzyme EcoRI (Fig. 5c), and the kanamycin resistance protein (Fig. 5e). We also expressed the extremely small fluorescent protein aptamer, FlAsH aptamer, which binds an arsenic ligand [28] (Fig. 5i). Thus, we covered a wide range of protein sizes, and many possible mRNA folds. ...
Background
Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits.
Results
Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates.
Conclusions
The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.
... Fluorophore haben im Vergleich zu fluoreszierenden Proteinen zusätzlich den Vorteil, in vivo sofort ohne eine Reifungszeit einsetzbar zu sein. Diese Aspekte finden sich analog in einer Weiterentwicklung der Proteinmarkierung mit Tetracystein wieder[87,88]. Die Tetracystein-Markierung von Proteinen basiert auf einem Fusionskonstrukt aus Protein und sechs Sulforhodamin B ist in Abbildung 4 zu sehen. ...
The RNase III-enzyme Dicer is involved in the posttranscriptional and translational regulation of gene expression.The human RNase Dicer shows, when purified, very low activity. Therefore, in this work, human Dicer was enzymatically characterized in cytosolic cell extracts in the presence of cytosolic cofactors. An enzyme assay based on changes of diffusion time was developed with fluorescence correlation spectroscopy (FCS). The enzyme substrate (ES)-complex did show a significantly higher diffusion time than product and substrate, and could therefore be distinguished from the two diffusion species. The dependence of enzyme activity on substrate concentration of human Dicer
was further investigated in cytosolic HEK293-cell extracts. The enzyme attained the half maximum enzyme activity at a substrate concentration of 76 nM. The pico- to femtomolar ES-complex concentration increased after Dicer overexpression and showed a similar dependence on substrate concentration as did enzyme activity. ES-complex concentration remained relatively constant during linear product formation. The analysis of the ES-complex
with respect to enzyme kinetic theory reveals the accuracy of the experimental data.
In a second project, the sulforhodamine B binding RNA aptamer SRB2m was analyzed by FCS. In this work it was demonstrated, that photophysical properties of the fluorophore as well as mobility and molecular size of the RNA in fluorophor-aptamer-complexes could be simultaneously measured by FCS. The sulforhodamine B-SRB2m complex showed a 4-fold
increased diffusion time compared to unbound sulforhodamine B. A second fluorophore, patent blue V (PBV), gave no autocorrelation function. The 36-fold increased molecular
brightness of the complex of PBV and SRB2m enabled FCS measurements with excellent signal-to-noise-ratio. The hydrodynamic radius rH of the PBV- and Sulforhodamin B-SRB2m
complex was 1.9 nm and 2.9 nm, respectively. The
hypothesis that a reduction of the molecular size of SRB2m by PBV due to a separation into monomers occurred, was verified by small angle X-ray scattering experiments.
... FlAsH/ReAsH is a biarsenical fluorophore that binds specifically to peptides containing amino acid sequences, such as CCXXCC, where X is an amino acid other than cysteine (Adams et al., 2002). Both FlAsH and ReAsH are highly permeable to cell membranes. ...
Light microscopy (LM) covers a relatively wide area and is suitable for observing the entire neuronal network. However, resolution of LM is insufficient to identify synapses and determine whether neighboring neurons are connected via synapses. In contrast, the resolution of electron microscopy (EM) is sufficiently high to detect synapses and is useful for identifying neuronal connectivity; however, serial images cannot easily show the entire morphology of neurons, as EM covers a relatively narrow region. Thus, covering a large area requires a large dataset. Furthermore, the three-dimensional (3D) reconstruction of neurons by EM requires considerable time and effort, and the segmentation of neurons is laborious. Correlative light and electron microscopy (CLEM) is an approach for correlating images obtained via LM and EM. Because LM and EM are complementary in terms of compensating for their shortcomings, CLEM is a powerful technique for the comprehensive analysis of neural circuits. This review provides an overview of recent advances in CLEM tools and methods, particularly the fluorescent probes available for CLEM and near-infrared branding technique to match LM and EM images. We also discuss the challenges and limitations associated with contemporary CLEM technologies.
... Caractérisation (Adams et al., 2002;Griffin et al., 1998). Cette approche est basée sur la forte affinité de l'arsenic (As) pour les groupements thiols retrouvés notamment sur les cystéines. ...
Les thérapies actuelles ciblant les étapes clés du cycle de réplication du VIH-1 sont puissantes et sélectives, mais des échecs thérapeutiques sont apparus en raison de l'émergence de résistance aux médicaments. Par conséquent, il existe un besoin urgent de développer de nouveaux médicaments qui utilisent des stratégies thérapeutiques alternatives. Cibler la protéine de nucléocapside (NCp7) est une stratégie prometteuse car NCp7 joue un rôle clé dans la réplication du VIH et sa séquence est hautement conservée dans les différentes souches du VIH. L’objectif du consortium européen THINPAD est de sélectionner des inhibiteurs spécifiques de la NCp7. Pour cribler les molécules, nous avons établi un test d’infectivité in cellulo utilisant la capacité des molécules sélectionnées à inhiber l'infection de cellules HeLa par des pseudo-particules non-réplicatives mimant la phase précoce du cycle d'infection virale du VIH-1. Afin de comprendre le mécanisme d'action des meilleures molécules sélectionnées, nous avons mis en place un test d'addition des molécules à différents temps post-infection et un test d'assemblage basé sur la technique de FRET-FLIM. La polyprotéine Gag est un précurseur nécessaire et suffisant pour la formation de particules virales mais le mécanisme de sélection de seulement deux copies d’ARN spécifique par virion est encore mal compris. Dans Gag, il y a deux sites d’interaction pour l'ARN. L’un de ces sites est le domaine de nucléocapside (NC), qui contient deux doigts de zinc capables de recruter l’ARN génomique viral. Le second site est constitué par les résidus basiques du domaine Matrice (MA). Ces deux sites peuvent interagir avec l'ARN simultanément et stabiliser la forme compacte de Gag. Pour démontrer in cellulo ce repliement de Gag, nous avons utilisé un système de split-Green Fluorescent Protein (GFP) et combiné différentes techniques de microscopie et de biochimie. Afin de compléter cette étude, le repliement sous forme compacte de différents mutants de Gag qui ont perdu totalement ou partiellement leurs capacités d’interaction avec l'ARN a également été étudié.
... Our objective was to incorporate the TC motif, CCPGCC (Adams et al. 2002;Griffin et al. 1998;Martin et al. 2005), into HO2 near each type of heme binding site (the catalytic core and the HRMs) to report on site-specific heme acquisition. A biarsenical indicator dye, FlAsH, binds to the TC motif and becomes fluorescent. ...
Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2. Our results indicate GAPDH and HO2 form a complex in vitro. We have followed heme insertion at both sites by fluorescence quenching in HEK293 cells with HO2 reporter constructs. Upon mutation of residues essential for heme binding at each site in our reporter construct, we found that HO2 binds heme at the core and the HRMs in live cells and that heme delivery to HO2 is dependent on the presence of GAPDH that is competent for heme binding. In sum, GAPDH is involved in heme delivery to HO2 but, surprisingly, not to a specific site on HO2. Our results thus emphasize the importance of heme binding to both the core and the HRMs and the interplay of HO2 with the heme pool via GAPDH to maintain cellular heme homeostasis.
... Advent of VCF with TMRM led to many studies that characterised conformational changes triggered by stimulation in several voltage-and ligand-gated ion channels (VGICs and LGICs, respectively), for instance: (1) Fluorescence signals underlying conformational changes associated with activation, inactivation and allosteric modulation of several VGICs [16,[18][19][20][21][22][23][24][25][26][27][28][29][30], (2) The effect of channel toxins and blockers binding on channel gating [31][32][33][34], (3) Characterisation of prokaryotic ion channels using single channel fluorescence spectroscopy [35][36][37] and (4) Characterisation of structural rearrangements on ligand binding in several LGICs that lead to channel modulation [38][39][40][41]. Other fluorophores that can be attached to cysteines of ion channels include bi-arsenic dyes FlAsH or ReAsH [42,43], organic dyes such as bimane [44,45] and photocross-linkers/photochromes [46,47]. Typical quenching measurements provide kinetics of the rearrangements occurring locally at the labelling site. ...
Ion channels undergo major conformational changes that lead to channel opening and ion conductance. Deciphering these structure-function relationships is paramount to understanding channel physiology and pathophysiology. Cryo-electron microscopy, crystallography and computer modelling provide atomic-scale snapshots of channel conformations in non-cellular environments but lack dynamic information that can be linked to functional results. Biophysical techniques such as electrophysiology, on the other hand, provide functional data with no structural information of the processes involved. Fluorescence spectroscopy techniques help bridge this gap in simultaneously obtaining structure-function correlates. These include voltage-clamp fluorometry, Förster resonance energy transfer, ligand binding assays, single molecule fluorescence and their variations. These techniques can be employed to unearth several features of ion channel behaviour. For instance, they provide real time information on local and global rearrangements that are inherent to channel properties. They also lend insights in trafficking, expression, and assembly of ion channels on the membrane surface. These methods have the advantage that they can be carried out in either native or heterologous systems. In this review, we briefly explain the principles of fluorescence and how these have been translated to study ion channel function. We also report several recent advances in fluorescence spectroscopy that has helped address and improve our understanding of the biophysical behaviours of different ion channel families.
Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.
Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis. Herein, we report a design strategy for probes that suppress undesired organelle accumulation using a bioisostere for intracellular protein imaging in living cells. Our design allows the protein labeling probes to possess both membrane permeability and suppress non-specific accumulation and has been shown to use several protein labeling systems, such as PYP-tag and Halo tag systems. We further developed a fluorogenic PYP-tag labeling probe for intracellular proteins and used it to visualize multiple localizations of target proteins in the intracellular system. Our strategy offers a versatile design for undesired accumulation-suppressed probes with cationic dye scaffolds and provides a valuable tool for intracellular protein imaging.
A split-protein system is a simple approach to introduce new termini which are useful as modification sites in protein engineering, but has been adapted mainly for monomeric proteins. Here we demonstrate the design of split subunits of the 60-mer artificial fusion-protein nanocage TIP60. The subunit fragments successfully reformed the cage structure in the same manner as prior to splitting. One of the newly introduced terminals at the interior surface can be modified using a tag peptide and green fluorescent protein. Therefore, the termini could serve as a versatile modification site for incorporating a wide variety of functional peptides and proteins.
Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures’ subcellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs’ primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon reaction with the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. Importantly, this was achieved by replacing only a fraction of GvpA with the tagged version. We used fluorescence images of the tagged GVs to study the GV size and distance distributions within these cells. This bioorthogonal and fractional labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.
Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
Bacterial secretion systems are among the largest protein complexes in prokaryotes and display remarkably complex architectures. Their assembly often follows clearly defined pathways. Deciphering these pathways not only reveals how bacteria accomplish to build these large functional complexes but can provide crucial information on the interactions and subcomplexes within secretion systems, their distribution within the bacterium, and even functional insights. Fluorescence microscopy provides a powerful tool for biological imaging, which presents an interesting method to accurately define the biogenesis of macromolecular complexes using fluorescently labeled components. Here, I describe the use of this method to decipher the assembly pathway of bacterial secretion systems.
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
Imaging proteins with high resolution is crucial for studying cellular physiology and pathology. Fluorescence imaging is a privileged method to visualize proteins with subcellular precision in live cells. In recent years, there has been a tremendous advance in the field of fluorescent dyes that are optically more sophisticated than genetically‐encodable fluorescent proteins. In this review, we aim to discuss modern bioconjugation methods to specifically incorporate these dyes into protein‐of‐interests. We focus on advances in live‐cell labeling strategies and fluorescent probes, especially the HaloTag, SNAP‐tag, TMP‐tag, and unnatural amino acid systems and their applications. These protein labeling methods, along with cutting‐edge dyes and novel microscopy methods, have become the infrastructure for biological research in the era of super‐resolution imaging.
Two non-canonical amino acids (ncAAs) with bio-orthogonal reactive groups, namely, p-azido-L-phenylalanine (p-AzF) and p-propargyloxy-L-phenylalanine (p-PaF), were genetically inserted into an aldo-keto reductase (AKR) and an alcohol dehydrogenase (ADH), respectively, at...
Delineating the complex network of interactions between antigen-specific T cells and antigen presenting cells (APCs) is crucial for effective precision therapies against cancer, chronic infections, and autoimmunity. However, the existing arsenal for examining antigen-specific T cell interactions is restricted to a select few antigen-T cell receptor pairs, with limited in situ utility. This lack of versatility is largely due to the disruptive effects of reagents on the immune synapse, which hinder real-time monitoring of antigen-specific interactions. To address this limitation, we have developed a novel and versatile immune monitoring strategy by adding a short cysteine-rich tag to antigenic peptides that emits fluorescence upon binding to thiol-reactive biarsenical hairpin compounds. Our findings demonstrate the specificity and durability of the novel antigen-targeting probes during dynamic immune monitoring in vitro and in vivo. This strategy opens new avenues for biological validation of T-cell receptors with newly identified epitopes by revealing the behavior of previously unrecognized antigen-receptor pairs, expanding our understanding of T cell responses.
Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid by Methanocaldococcus jannaschii tyrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNAPyl pairs from Methanosarcina mazei and Ca. Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons.
Here we describe the application of genetic code expansion and site-specific incorporation of noncanonical amino acids that serve as anchor points for fluorescent labeling to generate bioluminescence resonance energy transfer (BRET)-based conformational sensors. Using a receptor with an N-terminal NanoLuciferase (Nluc) and a fluorescently labeled noncanonical amino acid in the receptor’s extracellular part allows to analyze receptor complex formation, dissociation, and conformational rearrangements over time and in living cells. These BRET sensors can be used to investigate ligand-induced intramolecular (cysteine-rich domain [CRD] dynamics), but also intermolecular (dimer dynamics) receptor rearrangements. With the design of BRET conformational sensors based on the minimally invasive bioorthogonal labeling procedure, we describe a method that can be used in a microtiter plate format and can be easily adopted to investigate ligand-induced dynamics in various membrane receptors.Key wordsConformational biosensorsBioluminescence resonance energy transferG protein-coupled receptorsAmber codon suppressionNoncanonical amino acidBioorthogonal labelingMolecular dynamicsMicrotiter plate reader
Mammalian metallothioneins (MTs) are small Cys-rich proteins involved in Zn(II) and Cu(I) homeostasis. They bind seven Zn(II) ions in two distinct β- and α-domains, forming Zn3Cys9 and Zn4Cys11 clusters, respectively. After six decades of research, their role in cellular buffering of Zn(II) ions has begun to be understood recently. This is because of different affinities of bound ions and the proteins’ co-existence in variously Zn(II)-loaded Zn4-7MT species in the cell. To date, it has remained unclear how these mechanisms of action occur and how the affinities are differentiated despite the Zn(S-Cys)4 coordination environment being the same. Here, we dissect the molecular basis of these phenomena by using several MT2 mutants, hybrid protein, and isolated domains. Through a combination of spectroscopic and stability studies, thiol(ate) reactivity, and steered molecular dynamics (SMD), we demonstrate that both protein folding and thermodynamics of Zn(II) ion (un)binding significantly differ between isolated domains and the whole protein. Close proximity reduces the degrees of freedom of separated domains, making them less dynamic. It is caused by the formation of intra- and interdomain electrostatic interactions. The energetic consequence of domains connection has a critical impact on the role of MTs in the cellular environment, where they function not only as a zinc sponge but also as a zinc buffering system keeping free Zn(II) in the right concentrations. Any change of that subtle system affects the folding mechanism, zinc site stabilities, and cellular zinc buffer components.
The full power of fluorescence technique can be demonstrated in imaging and sensing within living cells. Its most important advantage that will probably be never competed by any other technique is a high spatial resolution together with ultimate contrast and sensitivitySensitivity obtained in a noninvasive or low invasive manner. Exploration of these possibilities has led to development of powerful methods of fluorescence microscopy, such as confocal, two-photonFluorescence microscopytwo-photon and evanescent-wave microscopy. Resolution in time and anisotropy were successfully applied to microscopy. Recent advances allowed achieving superresolution images and observing individual molecules.
Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA,a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.
Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression. Meanwhile, we developed a highly efficient droplet-based microfluidics system integrating the FlAsH-tetracysteine assay that enables screening of millions of strains to identify potential knockdowns conducive to nanobody VHH secretion. Among our highest-ranking candidates are a slew of previously unknown targets involved in transmembrane transport, amino-acid metabolism and redox regulation. Guided by these findings, we eventually constructed a hyperproducer for multiple proteins via combinatorial engineering of redox-response transcription factors. As the near-universal applicability of CRISPRi technology and the FlAsH-based screening platform, this procedure might be expanded to include a varied variety of microbial species and recombinant proteins.
The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.
Human β-defensin 3, HBD-3, is a 45-residue antimicrobial and immunomodulatory peptide that plays multiple roles in the host defense system. In addition to interacting with cell membranes, HBD-3 is also a ligand for melanocortin receptors, cytokine receptors and voltage-gated potassium channels. Structural and functional studies of HBD-3 have been hampered by inefficient synthetic and recombinant expression methods. Herein, we report an optimized Fmoc solid-phase synthesis of this peptide using an orthogonal disulfide bonds formation strategy. Our results suggest that utilization of an optimized resin, coupling reagents and pseudoproline dipeptide building blocks decrease chain aggregation and largely improve the amount of the target peptide in the final crude material, making the synthesis more efficient. We also present an alternative synthesis of HBD-3 in which a replacement of a native disulfide bridge with a diselenide bond improved the oxidative folding. Our work enables further biological and pharmacological characterization of HBD-3, hence advancing our understanding of its therapeutic potential.
Fluorescent probes are undoubtedly powerful tools for the study of living organisms. Here, we present the design of a hybrid chemogenetic reporter based on the FAST (Fluorescence-Activating and absorption Shifting Tag) system. We applied a coordinated molecular and protein engineering strategy to develop a small protein tag, pFAST, with suitable properties for an entire collection of fluorogenic chromophores, spanning the entire visible spectrum. The ability to adapt the fluorescence of a single tag by choosing a different live-cell compatible fluorogenic chromophore provides a large experimental versatility, allowing to image live cells and multicellular organisms using both conventional and super-resolution microscopy. Finally, we tailored pFAST for the manipulation of biological systems by the design of chemogenetic photosensitizers. Such photosensitizers are known to generate reactive oxygen species (ROS) which induce various biological processes in live cells upon light illumination.
Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature gamma-aminobutyric acid type-A (GABAA) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl-family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins and phosphoinositides to recruit gephyrin and GABAA receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin FRET sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction, which does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.
Vesicle-associated membrane protein (VAMP) 1 is a 120-amino acid protein which co-purifies with cholinergic synaptic vesicles from the marine ray Torpedo californica. We used the Torpedo gene to isolate two independent classes of VAMP cDNA clones from rat brain. Nucleotide sequence analysis of the cDNAs predicts proteins which are 84 and 75% homologous to Torpedo VAMP-1. The amino-terminal 24–28 amino acid residues which comprise the proline-rich head are only about 50% homologous between the different VAMPs, yet the proline-rich character is maintained. The 69 amino acids which comprise the hydrophilic core are highly homologous to Torpedo VAMP-1, with only 2 amino acid substitutions in rat VAMP-1 and 6 in rat VAMP-2. The carboxyl-terminal 23 amino acids of all of the VAMP proteins maintain the hydrophobic character necessary to serve as a membrane anchor. Both VAMP transcripts are expressed differentially in the rat central nervous system. Whereas VAMP-2 is more highly expressed in the whole brain, VAMP-1 is expressed at a higher level in the spinal cord.
Tris(vancomycin carboxamide) binds a trivalent ligand derived fromd-Ala-d-Ala with very high affinity: dissociation constant (K
d) ≈ 4 × 10–17 ± 1 × 10–17 M. High-affinity trivalent binding and monovalent binding are fundamentally different. In trivalent (and more generally,
polyvalent) binding, dissociation occurs in stages, and its rate can be accelerated by monovalent ligand at sufficiently high
concentrations. In monovalent binding, dissociation is determined solely by the rate constant for dissociation and cannot
be accelerated by added monomer. Calorimetric measurements for the trivalent system indicate an approximately additive gain
in enthalpy relative to the corresponding monomers. This system is one of the most stable organic receptor-ligand pairs involving
small molecules that is known. It illustrates the practicality of designing very high-affinity systems based on polyvalency.
Using synthetic oligonucleotides as probes we have cloned the streptavidin gene from a genomic library of Streptomyces avidinii. Nucleotide sequence analysis indicated that a 2 Kb DNA-fragment contained the entire coding region, a signal peptide region
and the 3′ and 5′ flanking regions of the gene. The deduced amino acid sequence shows several interrupted blocks of homology
with the amino acid sequence of chicken egg-white avidin. Analysis of the secondary structure suggests a high content of beta-structure
in both proteins and considerable overall structural similarity between them.
A specific and sensitive assay for disulfide groups has been developed, based on reduction with dithioerythritol or dithiothreitol and determination of the resulting monothiols with 5,5′-dithiobis(2-nitrobenzoic acid) in the presence of arsenite. The dissociation constants for the complexes formed between arsenite and dithioerythritol, dithiothreitol, 1,3-dithioglycerol, and 1,2-dithioglycerol have been measured, as well as the rate constants for the formation and breakdown of the complexes and for the bimolecular reaction between the complexes and 5,5′-dithiobis(2-nitrobenzoic acid). The pK values of dithiothreitol are 8.3 and 9.5, and those for dithioerythritol are 9.0 and 9.9.
A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.
Expansion of DNA trinucleotide repeats (TNRs) is the causative mutation in a growing number of human genetic diseases. Large expansions of a CTG tract were obtained and shown by genetic and physical assays to be length-dependent sites of chromosome breakage in Saccharomyces cerevisiae. Deletion of RAD27, which encodes a nuclease involved in Okazaki fragment processing, caused length-dependent destabilization of CTG tracts and a substantial increase in expansion frequency. The genetic assay described here can be used to evaluate other factors that induce TNR expansion or chromosome fragility in humans.
Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo.
A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here 'fluorettes', was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat pIII protein of the filamentous bacteriophage M13. The 'best' fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red. Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution. In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye.
Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection. Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes. Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.
Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.
A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones.
We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing
avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pHER = 7.4 ± 0.2, mean ± S.D.) to Golgi (pHG = 6.2 ± 0.4) to mature secretory granules (MSGs) (pHMSG= 5.5 ± 0.4). Golgi and MSGs required active H+v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H+ leak rates across each membrane. However, neither steady-state pHMSG nor rates of passive H+ leak were affected by Cl−-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pHMSG. Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl− conductances. Measurements of H+ leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical
model to determine the H+ permeability (P
H+) of each organelle membrane. We found that P
H+ decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases inP
H+ and successive increases in the active H+ pump density.
Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.
The condensation of resorcinol and 4-chloro resorcinol with chloro-and nitro-substituted β-resorcylic acids, has yielded the corresponding tetrahydroxy benzophenones, which with the exception of those containing nitro groups, could be cyclised to the respective 3:6-dihydroxy xanthones. In the case of condensations with orcinol, however, 1:6-dihydroxy xanthones could be directly isolated. A number of halo and nitro derivatives of 3:6-dihydroxy xanthone have also been prepared by direct halogenation and nitration of the xanthone, as well as by substitution in the intermediate benzophenone and subsequent cyclisation.
Recombinant proteins containing four cysteines at the i,i + 1, i + 4, and i + 5 positions of an α helix were fluorescently labeled in living cells by extracellular administration of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein.
This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the
tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility
in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly
modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently
stained by small nonfluorescent dye molecules added from outside the cells.
The cooperativity between binding of cell wall precursor analogs (ligands) to and antibiotic dimerization of the clinically
important vancomycin group antibiotics was investigated by nuclear magnetic resonance. When dimerization was weak in the absence
of a ligand, the increase in the dimerization constant in the presence of a ligand derived largely from changes associated
with tightening of the dimer interface. When dimerization was strong in the absence of a ligand, the increase in the dimerization
constant in the presence of a ligand derived largely from changes associated with tightening of the ligand-antibiotic interface.
These results illustrate how, when a protein has a loose structure, the binding energy of another molecule to the protein
can derive in part from changes occurring within the protein.
Hydroxy-xanthones and -benzophenones are conveniently obtained from hydroxybenzoic acids and phenols in presence of zinc chloride and phosphorus oxychloride.
Uranine and its thioxanthene and selenoxanthene analogues, as well as the disodium salts of o-(6- hydroxy-3-oxo-3H-xanthen-9-yl)benzenesulphonic acid and its thioxanthene and selenoxanthene analogues, show different photosensitizer properties in the E/Z-isomerization of trienes. This has been verified by applying them to the technically useful conversion of tachysterol into previtamin D. The syntheses of the novel photosensitizers with thioxanthene and selenoxanthene moieties are also described.
A simple two-armed receptor has been prepared that binds certain peptides sequence-selectively in water at pH 4.
The introduction of immobilized metal ion affinity chromatography, directed toward specific protein side chains, has opened a new dimension in protein purification. This review covers the principles and practice of IMAC that can be performed under very mild, nondenaturing conditions. IMAC is particularly suitable for preparative group fractionation of complex extracts and biofluids, but can also be used in high-performance mode: "HP-IMAC." Single-step purifications of 1000-fold or more may allow isolation of a particular protein from crude extracts on a milligram or gram scale. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography, and the immobilized metal ion ligand complexes are more likely to withstand wear and tear than are antibodies or enzymes. The enormous potential of IMAC and related metal affinity techniques is only in the initial stages of being explored and exploited. Synthesis of IMA adsorbents, and various modes of performing IMAC are discussed and exemplified with selected applications. Advantages and disadvantages are listed. Effective means of counteracting the few undesirable effects that can occur are suggested.
We describe the construction of systems for expressing the cloned streptavidin gene in Escherichia coli. Although the streptavidin gene is extremely lethal to the host cells, because of the strong biotin binding of the gene product, the gene was expressed efficiently in E. coli by using T7 RNA polymerase/T7 promoter expression systems. The expressed streptavidin accumulated to more than 35% of the total cell protein. The expressed streptavidin was insoluble in the cell. However, after solubilization by dialysis against 6 M guanidine hydrochloride (pH 1.5) and removal of guanidine hydrochloride by dialysis, the protein became soluble and renatured. This simple procedure yielded streptavidin purified almost to homogeneity. The purified streptavidin bound 3.5-3.9 molecules of biotin per molecule, indicating that it had almost full biotin-binding ability. Some of the purified streptavidin molecules aggregated into oligomers, suggesting that the C-terminal region of the molecule, present in our material but absent in typical preparations, may be responsible for the aggregation.
The contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated on this technique using actin, myosin and the actomyosin complex. These papers are reviewed with several aims in mind: (i) we assess the reliability and consistency of intra- and inter-molecular distances measured between the fluorescent probes attached to specific sites on these proteins; (ii) we determine whether the measurements can be assembled into an internally consistent model which can be fitted to the known dimensions of the actomyosin complex; (iii) several of the FRET distances are consistent with the available structural data from crystallographic and electron microscopic dimensions; (iv) the modelled FRET distances suggest that the assumed value of the orientation factor (K
2 = 2/3) is reasonable; (v) we conclude that the model has a predictive value, i.e. it suggests that a small number of the published dimensions may be incorrect and predicts the magnitude of a larger number of measurements which have not yet been reported; and finally (vi) we discuss the contribution of FRET determinations to the current debate on the molecular mechanism of contraction.
Static and dynamic measurements of fluorescence anisotropy have been made for calmodulin, employing both the intrinsic fluorescence of Tyr-99 and Tyr-138 and the fluorescence of dansyl and fluorescein groups attached to Tyr-99, as well as AEDANS groups attached to methionines. All approaches indicate the presence of a significant internal mobility involving the probe for calmodulin in the absence of Ca2+. This is diminished in the presence of Ca2+.2 2 This research was supported by NIH Grant AM 30322.
Native proteins with cysteine-sulfhydryls in proximity that can undergo reversible dithiol/disulfide conversions are here referred to as vicinal dithiol-containing proteins (VDPs). The dithiols of interest are present at the protein surface, allowing interaction with substrates, with oxidants, and with proteins catalyzing disulfide reduction. VDPs include enzymes whose active sites contain redox-active dithiols, such as nucleotide and oxidized glutathione (GSSG) reductases, thioredoxin reductase-thioredoxin, and glutaredoxin. Additionally, proteins that bind to DNA including transcription regulatory proteins with zinc fingers and most polymerases are VDPs. Also included are a series of enzymes in which the dithiol/disulfide conversion serves as an allosteric regulator of the enzymatic activity. Hormone or steroid receptors, such as the triiodothyronine nuclear receptor and the cytoplasmic glucocorticoid receptor have a dithiol that regulates hormone or steroid binding to the receptor and subsequent binding of the receptor to DNA. Another distinctive property of VDPs is their high affinity for trivalent arsenicals. This interaction is stable in the absence of other dithiols. On addition of small dithiols, such as 1,2-dithioglycerol (BAL), dithiothreitol, or lipoic acid, however, the binding of the arsenical to the dithiol protein can be reversed.
Proteins containing vicinal dithiols were purified by affinity chromatography using Sepharose 4B linked to aminohexanoyl-4-aminophenylarsineoxide (As-Sepharose). The protein vicinal dithiols form stable dithioarsine derivatives with the arsine oxide moieties of the gel. The adsorbed proteins were eluted, at physiological pH, by buffers containing beta-mercaptoethanol or dithiothreitol. The dithiol proteins were identified by their specific labeling with N-iodoacetyl-3-[125I]-iodotyrosine. Cytoplasmic thiol proteins of L1210 murine leukemia lymphoblasts were separated into three classes by interaction with As-Sepharose. Proteins that did not bind to the gel consisted of monothiol proteins; proteins eluted by beta-mercaptoethanol include vicinal dithiol-containing proteins with low affinity for the arsine oxide. DL-Dithiothreitol (DTT) elutes a large group of vicinal dithiol-containing proteins with high affinity for the arsine groups. Gradient elution allowed characterization of the relative affinities of dithiol proteins for the As-Sepharose. A one-step purification of the L-triiodothyronine recombinant rat c-erb A beta 1 T3 receptor synthesized in yeast required pretreatment with DTT for binding to As-Sepharose and resulted in a 62-fold increase in specific activity. The procedure allows purification of proteins inhibited by phenylarsine oxide such as phosphotyrosine phosphatases, proteins that are subject to redox regulation, and dithiol proteins that are targets of oxidative stress.
A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
To determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow-green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488-nm excitation, a rapid cut-on dichroic mirror and a narrow-bandpass emission filter. Two-photon excitation of GFP fluorescence at the equivalent of approximately 390 nm provided better absorption than did 488-nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione-S-transferase (GST) or with glucokinase. Furthermore, purified GST.GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than approximately 1 microM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane, organelles or nucleus.
An efficient preparative method for separating 5- and 6-carboxyfluorescein is presented. 6-Carboxyfluorescein dipivalate is isolated as its diisopropylamine salt, which can be converted to the free acid or used directly in coupling reactions. The 5-isomer is isolated from the acidified mother liquor. Isomerically pure carboxyfluoresceins are prepared by hydrolysis of the corresponding dipivalates.
In just three years, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. High-resolution crystal structures of GFP offer unprecedented opportunities to understand and manipulate the relation between protein structure and spectroscopic function. GFP has become well established as a marker of gene expression and protein targeting in intact cells and organisms. Mutagenesis and engineering of GFP into chimeric proteins are opening new vistas in physiological indicators, biosensors, and photochemical memories.
Mammalian organelles of the secretory pathway are of differing pH. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still ( approximately pH 5). The mechanisms that regulate pH in these organelles are still unknown.
Using a novel method, we tested whether differences in H(+) 'leak' and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. A pH-sensitive, membrane-permeable fluorescein-biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. In live, intact cells, ER pH (pH(ER)) was 7.2 +/- 0.2 and Golgi pH (pH(G)) was 6.4 +/- 0.3 and was dissipated by bafilomycin. Buffer capacities of the cytosol, ER and Golgi were all similar (6-10 mM/pH). ER membranes had an apparent H(+) permeability three times greater than that of Golgi membranes. Removal of either K(+) or Cl(-) did not affect ER and Golgi H(+) leak rates, or steady-state pH(G) and pH(ER).
The Golgi is more acidic than the ER because it has an active H(+) pump and fewer or smaller H(+) leaks. Neither buffer capacity nor counterion permeabilities were key determinants of pH(G), pH(ER) or ER/Golgi H(+) leak rates.
Genetically-encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269-272). We find that kinesin tagged with this cysteine-containing helix binds specifically to FlAsH resin and can be eluted in a fully active form. This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin. Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol. Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.
The ubiquitin (Ub) fusion technique was developed through experiments in which a segment of DNA encoding the 76-residue Ub was joined to DNA encoding Escherichia coli β-galactosidase (βgal). A major application of the Ub fusion technique is its use to augment the yields of recombinant proteins. This approach increases the yield of short peptides as well. The Ub-mediated increase in total yield is often accompanied by an even greater increase in the solubility of overexpressed protein. The Ub fusion technique is made possible by the ability of deubiquitylating enzymes (DUBs) to cleave a Ub fusion in vivo or in vitro after the last residue of Ub irrespective of the flanking sequence context. The Ub fusion technique has given rise to a number of applications whose common feature is utilization of the rapid and highly UPR (ubiquitin/protein/reference) technique that increases the accuracy of pulse-chase and analogous measurements. The Ub sandwich technique, a descendant of UPR, has made it possible to determine the extent of cotranslational protein degradation in vivo for any protein of interest.
This chapter describes how the avidin chimera proteins are expressed in specific organelles of mammalian cells, the synthesis and characteristics of the Flubida dyes, and the methods used for labeling avidin-containing compartments with Flubi dyes and then studying pH regulation in mammalian organelles. Avidin chimera proteins are localized to specific compartments by appending organelle-specific targeting sequences to avidin. Cells expressing the organelle-specific avidin chimera proteins have been loaded with Flubida-2, a membrance-permeable, pH-sensitive fluorescein-biotin derivative. The targeted avidin-Flubi approach is advantageous because it allow specific targeting of a ratiometric pH-sensitive dye to the ER and Golgi of mammalian cells. The method provides flexibility in that the Flubi dyes can be modified chemically to provide different pH sensitivities that would be suitable for making measurements in both neutral organelles (e.g., ER) as well as in organelles that may vary in acidity from pH 6.4 (e.g., Golgi) to pH 4.5 (lysosomes).
DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming C==N==C==O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C==N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C==N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.
DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.
Chimeric RNA/DNA oligonucleotides have been used successfully to correct point and frameshift mutations in cells as well as in animal and plant models. This approach is one of several nucleic acid repair technologies that will help elucidate the function of newly discovered genes. Understanding the mechanisms by which these different technologies direct gene alteration is essential for progress in their application to functional genomics.
We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.
FLASH-EDT2--4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2--has been reported to fluoresce only after binding with high affinity to a specific tetracysteine motif (CCXXCC, "Cys4") and thus to provide a technique for labeling recombinant proteins in vivo (Griffin et al. Science 281:269-272). We have attempted to use FLASH-EDT2 as a site-specific label of the II-III loop of the dihydropyridine receptor (DHPR) in skeletal muscle. Upon expression in dysgenic myotubes (which lack endogenous alpha1s), an alpha1s mutated to contain CCRECC in the II-III loop was able to produce L-type calcium currents and to mediate skeletal-type excitation-contraction (EC) coupling, but FLASH-EDT2 labeling revealed no difference from non-transfected dysgenic myotubes. HeLa-S3 cells transfected with Cys4-containing calmodulin were significantly more fluorescent than non-transfected cells, whereas the difference between transfected and non-transfected cells was less apparent for CHO-K and HEK 293 cells. Because the fluorescence of non-transfected cells increased substantially after treatment with FLASH-EDT2, it suggested the possibility that FLASH binds to endogenous cysteine-containing proteins. This finding was confirmed in cuvette experiments in which FLASH-EDT2 fluorescence was observed after FLASH-EDT, was added to protein homogenates from myotubes or cell lines. The enhanced fluorescence was abolished by pretreatment of cells or cell homogenates with coumarine maleimide (CPM), which modifies cysteine residues covalently. Thus, enhanced FLASH fluorescence appears to occur both after binding to an introduced Cys4 motif and to endogenous, cysteine-containing proteins. Therefore, FLASH-EDT2 may be useful only for labeling those recombinant proteins that express at a very high level.
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