Bulky Protein Research Articles

Out-of-equilibrium mechanochemistry and self-organization of fluid membranes interacting with curved proteins

The function of biological membranes is controlled by the interaction of the fluid lipid bilayer with various proteins, some of which induce or react to curvature. These proteins can preferentially bind or diffuse towards curved regions of the membrane, induce or stabilize membrane curvature and sequester membrane area into protein-rich curved domains. The resulting tight interplay between mechanics and chemistry is thought to control organelle morphogenesis and dynamics, including traffic, membrane mechanotransduction, or membrane area regulation and tension buffering. Despite all these processes are fundamentally dynamical, previous work has largely focused on equilibrium and a self-consistent theoretical treatment of the dynamics of curvature sensing and generation has been lacking. Here, we develop a general theoretical and computational framework based on a nonlinear Onsager’s formalism of irreversible thermodynamics for the dynamics of curved proteins and membranes. We develop variants of the model, one of which accounts for membrane curving by asymmetric crowding of bulky off-membrane protein domains. As illustrated by a selection of test cases, the resulting governing equations and numerical simulations provide a foundation to understand the dynamics of curvature sensing, curvature generation, and more generally membrane curvature mechano-chemistry.

An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist

cGMP signaling is one of the master regulators of diverse functions in eukaryotes; however, its architecture and functioning in protozoans remain poorly understood. Herein, we report an exclusive guanylate cyclase coupled with N-terminal P4-ATPase in a common parasitic protist, Toxoplasma gondii This bulky protein (477-kD), termed TgATPaseP-GC to fairly reflect its envisaged multifunctionality, localizes in the plasma membrane at the apical pole of the parasite, whereas the corresponding cGMP-dependent protein kinase (TgPKG) is distributed in the cytomembranes. TgATPaseP-GC is refractory to genetic deletion, and its CRISPR/Cas9-assisted disruption aborts the lytic cycle of T. gondii Besides, Cre/loxP-mediated knockdown of TgATPaseP-GC reduced the synthesis of cGMP and inhibited the parasite growth due to impairments in the motility-dependent egress and invasion events. Equally, repression of TgPKG by a similar strategy recapitulated phenotypes of the TgATPaseP-GC-depleted mutant. Notably, despite a temporally restricted function, TgATPaseP-GC is expressed constitutively throughout the lytic cycle, entailing a post-translational regulation of cGMP signaling. Not least, the occurrence of TgATPaseP-GC orthologs in several other alveolates implies a divergent functional repurposing of cGMP signaling in protozoans, and offers an excellent drug target against the parasitic protists.

Structural Optimization of α-Synuclein Fibril Growth Inhibitors

Aggregation of alpha-synuclein (αSyn) is involved in the development of Parkinson's disease, a severe neurodegenerative disorder that has no cure at the moment. Recently we showed that bulky molecules binding to the ends of αSyn fibrils can efficiently slowdown fibril growth and developed the first rationally designed inhibitor working on this principle [1]. The constructed fusion protein, consisting of a bulky protein moiety attached to the C-terminus of αSyn molecule, has IC50 of 850 nM. Nevertheless, its inhibitor efficiency can be further improved by increasing steric hindrance and affinity to the fibril end. We studied the structure-activity relationship in a series of inhibitors focusing on the structure, size, and position of the bulky group. We prepared 12 protein-based inhibitors varying: a) sequence of αSyn moiety to improve binding of inhibitor to the fibril end; b) structure of bulky moieties; c) attachment position of the bulky group to αSyn to improve steric hindrance. Their inhibition activities were tested in a kinetic ThT-based assay and compared by IC50 values. Decreasing the electrostatic repulsion between the fibril end and inhibitor molecule by deleting αSyn C-terminus or neutralizing its charge significantly improved inhibitor efficiency (IC50=500 nM and 700 nM, respectively). Changing the bulky group structure and positioning it at the N-terminus allowed us to prepare an inhibitor with IC50 340 nM, that is the most active αSyn fibril growth inhibitor to the best of our knowledge. Our structure-activity relationship studies provide background for rational design of protein-based inhibitors of αSyn fibrillization targeting ends of fibrils and having potential to reach low nanomolar IC50. [1] V V Shvadchak, K Afitska, D A Yushchenko, Angew Chem Int Ed Engl, 2018, 57, 5690.

Size-Selective Hydroformylation by a Rhodium Catalyst Confined in a Supramolecular Cage.

Size‐selective hydroformylation of terminal alkenes was attained upon embedding a rhodium bisphosphine complex in a supramolecular metal–organic cage that was formed by subcomponent self‐assembly. The catalyst was bound in the cage by a ligand‐template approach, in which pyridyl–zinc(II) porphyrin interactions led to high association constants (>105 m −1) for the binding of the ligands and the corresponding rhodium complex. DFT calculations confirm that the second coordination sphere forces the encapsulated active species to adopt the ee coordination geometry (i.e., both phosphine ligands in equatorial positions), in line with in situ high‐pressure IR studies of the host–guest complex. The window aperture of the cage decreases slightly upon binding the catalyst. As a result, the diffusion of larger substrates into the cage is slower compared to that of smaller substrates. Consequently, the encapsulated rhodium catalyst displays substrate selectivity, converting smaller substrates faster to the corresponding aldehydes. This selectivity bears a resemblance to an effect observed in nature, where enzymes are able to discriminate between substrates based on shape and size by embedding the active site deep inside the hydrophobic pocket of a bulky protein structure.

Polyamine Deacetylase Structure and Catalysis: Prokaryotic Acetylpolyamine Amidohydrolase and Eukaryotic HDAC10.

Polyamines such as putrescine, spermidine, and spermine are small aliphatic cations that serve myriad biological functions in all forms of life. While polyamine biosynthesis and cellular trafficking pathways are generally well-defined, only recently has the molecular basis of reversible polyamine acetylation been established. In particular, enzymes that catalyze polyamine deacetylation reactions have been identified and structurally characterized: histone deacetylase 10 (HDAC10) from Homo sapiens and Danio rerio (zebrafish) is a highly specific N8-acetylspermidine deacetylase, and its prokaryotic counterpart, acetylpolyamine amidohydrolase (APAH) from Mycoplana ramosa, is a broad-specificity polyamine deacetylase. Similar to the greater family of HDACs, which mainly serve as lysine deacetylases, both enzymes adopt the characteristic arginase-deacetylase fold and employ a Zn2+-activated water molecule for catalysis. In contrast with HDACs, however, the active sites of HDAC10 and APAH are sterically constricted to enforce specificity for long, slender polyamine substrates and exclude bulky peptides and proteins containing acetyl-l-lysine. Crystal structures of APAH and D. rerio HDAC10 reveal that quaternary structure, i.e., dimer assembly, provides the steric constriction that directs the polyamine substrate specificity of APAH, whereas tertiary structure, a unique 310 helix defined by the P(E,A)CE motif, provides the steric constriction that directs the polyamine substrate specificity of HDAC10. Given the recent identification of HDAC10 and spermidine as mediators of autophagy, HDAC10 is rapidly emerging as a biomarker and target for the design of isozyme-selective inhibitors that will suppress autophagic responses to cancer chemotherapy, thereby rendering cancer cells more susceptible to cytotoxic drugs.

Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectroscopy

In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.

Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen-substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH-EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.-Bohl, C., Pomorski, A., Seemann, S., Knospe, A.-M., Zheng, C., Krężel, A., Rolfs, A., Lukas, J. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers.

Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors.

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549).

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

Abstract PR10: A glycoprotein-mediated mechanical switch promotes glioma aggression

Abstract Glioblastoma multiforme (GBM) is a malignant brain tumor whose progression is associated with rampant extracellular matrix (ECM) remodeling. We recently found that ECM stiffness correlates with poor survival in human GBM specimens. Glycoproteins are the major constituent of normal brain ECM and many are overexpressed in brain tumors, yet the interplay between glycoproteins and mechanical signaling in GBM pathogenesis remains poorly understood. Here, we show that bulky glycoproteins and sugar-binding proteins are broadly upregulated in GBM relative to lower grade gliomas. Further, these genes are overexpressed in the mesenchymal (Mes) relative to the proneural (Pro) GBM subclass, the former of which is associated with treatment resistance and relapse. We took a specific interest in the hyaluronic acid (HA)-producing enzyme, HAS2, and the galactoside-binding lectin galectin-1 (Gal1) due to their ability to modulate tissue structure and rheology. Using mouse models of human GBM we showed that Mes tumors are enriched in HA and fibronectin, coincident with elevated ECM stiffness and mechanical signaling. These data suggest the possibility that aberrant glycoprotein expression drives GBM aggression through enhanced mechanical signaling resulting from tissue stiffening. Consistent with this hypothesis, by elevating mechanical signaling in Pro GBMs we induce a robust Mes-like transition and we see the opposite when reducing Gal1 expression or HA content in Mes tumors. Our data provides evidence of a feed-forward mechanism whereby mechanical signaling drives Gal1 and HA production which reinforce ECM stiffness, thus sustaining pro-tumorigenic mechanical signaling. This abstract is also presented as Poster A21. Citation Format: J Matthew Barnes, Yekaterina A. Miroshnikova, Jason C. Tung, Russel O. Bainer, Valerie M. Weaver. A glycoprotein-mediated mechanical switch promotes glioma aggression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR10.

Quantification of the degree of biotinylation of proteins using proteinase K digestion and competition ELISA

Quantification of the degree of biotinylation of proteins is useful to achieve and maintain a high degree of consistency of reagents used in research and diagnostic setting. Unfortunately, existing protocols and commercial kits suffer from a number of shortcomings that limit their usefulness. Here, we describe a simple protocol that overcomes the limitations of current assays. A robust competition ELISA was developed that is easy to carry out, uses no specialized equipment other than a standard plate reader for absorbance measurements and only reagents that are commonly available. The protocol uses a proteinase K digestion step of a sample of biotinylated protein to eliminate multivalency issues and sterical hindrance from bulky proteins. Furthermore, the use of an anti-biotin antibody instead of streptavidin results in a convenient range of sensitivity, avoiding million-fold dilutions that may impair precision. The resulting assay typically consumes about 1μgof biotinylated protein.

Crowding of receptors induces ring-like adhesions in model membranes

The dynamics of formation of macromolecular structures in adherent membranes is a key to a number of cellular processes. However, the interplay between protein reaction kinetics, diffusion and the morphology of the growing domains, governed by membrane mediated interactions, is still poorly understood. Here we show, experimentally and in simulations, that a rich phase diagram emerges from the competition between binding, cooperativity, molecular crowding and membrane spreading. In the cellular context, the spontaneously-occurring organization of adhesion domains in ring-like morphologies is particularly interesting. These are stabilized by the crowding of bulky proteins, and the membrane-transmitted correlations between bonds. Depending on the density of the receptors, this phase may be circumvented, and instead, the adhesions may grow homogeneously in the contact zone between two membranes. If the development of adhesion occurs simultaneously with membrane spreading, much higher accumulation of binders can be achieved depending on the velocity of spreading. The mechanisms identified here, in the context of our mimetic model, may shed light on the structuring of adhesions in the contact zones between two living cells. This article is part of a Special Issue entitled: Mechanobiology.

Targeting Aspects of Nanogels: An Overview

Nanogels have shown a great potential for the delivery of large number of drugs to different organs of the body owing to their high biocompatibility, high drug loading capacity, high biodegradability (and hence low cytotoxicity), good permeation capabilities and tissue mimicking properties. Their high water retention makes them ideal capable of incorporation of bulky drugs like proteins, peptides, oligonucleotides and other macromolecules. All these properties of nanogels make them able to carry number of drugs to vast number of organs. Nanogels have shown potential in many fields including chemotherapy, diagnosis, organ tergeting, gene delivery and many others. The main areas of the target for the nanogels have been tumors of brain, liver, skin etc. Other uses of the nanogel are in diabetes, inflammation, wound healing, local anesthesia etc. This review concentrates over the targeting potential of nanogels in different organs for various conditions.

Hierarchical porous materials based on nanoscale metal-organic frameworks dominated with permanent interparticle porosity

In order to extensively synthesize mesoporous metal-organic frameworks (mesoMOFs), we developed a strategy to construct permanent interparticle porosity based on nanoscale metal-organic frameworks (NMOFs). Using the strategy, we have attained a series of interparticle porosity dominated mesoMOFs (IPD-mesoMOFs) from six MOF-types with MIl-100, MIL-53, HKUST-1, DUT-5, DUT-4 and MIL-101(Cr) (termed as IPD-mesoMOF-1, -2, -3, -4, -5 and -6) structure, respectively. All members of this series are not only comparable to inorganic mesoporous materials to have the tunable mesopore apertures varying from a few nanometers to over a dozen nanometers, but also superior to the inorganic counterparts to remain hierarchical porosity with higher surface area (up to 2130m2g−1), larger mesopore volume (the highest to 2.59cm3g−1) and optional micro-porosity of diverse crystalline structures. The large mesopore apertures and rich carboxyl residues on the mesopore-walls also allow the IPD-mesoMOF series to accommodate large organic and inorganic molecules, especially to immobilize the bulky natural protein, such as, hemoglobin.

Label‐Free Impedance Aptasensor for Major Peanut Allergen Ara h 1

AbstractThe peanut allergen Ara h 1 belonging to the vicilin family of seed storage proteins is known to be a suitable marker for peanut detection in foods. In the present work, we suggest an electrochemical detection of Ara h 1 using a recently selected 80‐base DNA aptamer. The detection strategy relies on insulation effect of a bulky protein captured by an immobilized aptamer. The electrodes were modified by the aptamer and characterized using electrochemical impedance spectroscopy (EIS). The aptamer surface density and analytical parameters of the calibration curves for Ara h 1 were compared with the system prepared with thrombin binding aptamer (TBA) and operated in the same conditions.

A Fractal Nature for Polymerized Laminin

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.

Protein imprinting over magnetic nanospheres via a surface grafted polymer for specific capture of hemoglobin

Combining surface molecular imprinting with a nano-sized physical form is an effective technology to achieve a large binding capacity and overcome template removal within molecularly imprinted polymers (MIPs), and is in particular advantageous to the template of bulky structures like proteins. In addition, the surface protein-imprinted polymers immobilized on nanospheres with magnetic function are more attractive in the research of selective recognition and enrichment of proteins. In this work, we have demonstrated a facile method, enhancing specific recognition of multifunctional molecularly imprinted magnetic nanospheres with core–shell structure. It was achieved simply via radical induced graft polymerization of low concentration monomers on the surface of vinyl groups modified magnetic nanospheres dispersed in aqueous media using hemoglobin (Hb) as a template. The possible gelation of the dispersion after polymerization was avoided and hence the imprinted magnetic particles without an obvious agglomerated phenomenon could be collected readily and expediently. Finally, a highly monodisperse magnetic nanospheres were synthesized successfully. The morphological studies of the resultant MIPs-coated magnetic particles were characterized using various methods. In batch rebinding tests, the imprinted magnetic particles reached saturated adsorption within 30 min and exhibited significant specific recognition towards the target protein molecules with a binding capacity as high as 281.94 mg g−1 and an imprinting factor of 4.29. The resultant Hb-MIPs magnetic particles not only extracted target protein from mixed proteins, but also enriched the low abundance of hemoglobin. Moreover, the stability and regeneration were also investigated, which indicated the excellent property of reutilization.

Monitoring Endocytic Trafficking of Anthrax Lethal Factor by Precise and Quantitative Protein Labeling

Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site-specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF's membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild-type LF. This allowed time-lapse imaging of LF's natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies.

Monitoring Endocytic Trafficking of Anthrax Lethal Factor by Precise and Quantitative Protein Labeling

AbstractCoupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site‐specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF’s membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild‐type LF. This allowed time‐lapse imaging of LF’s natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies.