Shiga Toxin B-subunit Research Articles

Engineered Synthetic STxB for Enhanced Cytosolic Delivery.

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8+ T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy.

In recent years, receptor-mediated drug delivery has gained major attention in the treatment of cancer. The pathogen-derived Shiga Toxin B subunit (STxB) can be used as a carrier that detects the tumor-associated glycosphingolipid globotriaosylceramide (Gb3) receptors. While drug conjugation via lysine or cysteine offers random drug attachment to carriers, click chemistry has the potential to improve the engineering of delivery systems as the site specificity can eliminate interference with the active binding site of tumor ligands. We present the production of recombinant STxB in its wild-type (STxBwt) version or incorporating the noncanonical amino acid azido lysine (STxBAzK). The STxBwt and STxBAzK were manufactured using a growth-decoupled Escherichia coli (E. coli)-based expression strain and analyzed via flow cytometry for Gb3 receptor recognition and specificity on two human colorectal adenocarcinoma cell lines-HT-29 and LS-174-characterized by high and low Gb3 abundance, respectively. Furthermore, STxBAzK was clicked to the antineoplastic agent monomethyl auristatin E (MMAE) and evaluated in cell-killing assays for its ability to deliver the drug to Gb3-expressing tumor cells. The STxBAzK-MMAE conjugate induced uptake and release of the MMAE drug in Gb3-positive tumor cells, reaching 94% of HT-29 cell elimination at 72 h post-treatment and low nanomolar doses while sparing LS-174 cells. STxBAzK is therefore presented as a well-functioning drug carrier, with a possible application in cancer therapy. This research demonstrates the feasibility of lectin carriers used in delivering drugs to tumor cells, with prospects for improved cancer therapy in terms of straightforward drug attachment and effective cancer cell elimination.

High-Relaxivity Molecular MRI Contrast Agent to Target Gb3-Expressing Cancer Cells.

Targeted contrast agents (CAs) can improve magnetic resonance imaging (MRI) for accurate cancer diagnosis. In this work, we used the Shiga toxin B-subunit (STxB) as a targeting agent, which binds to Gb3, a glycosphingolipid highly overexpressed on the surface of tumor cells. We developed STxB-targeted MRI probes from cyclic peptide scaffolds functionalized with six to nine monoamide DO3A[Gd(III)] chelates. The influence of structural constraints on the longitudinal relaxivity (r1) of the CAs has been studied. The cyclic peptide carrying nine monoamide DO3A[Gd(III)] exhibited a r1 per compound of 32 and 93 mM-1s-1 at 9.4 and 1.5 T, respectively. Its conjugation to the pentameric STxB protein led to a 70 kDa compound with a higher r1 of 150 and 475 mM-1 s-1 at 9.4 and 1.5 T, respectively. Specific accumulation and cellular distribution of this conjugate in Gb3-expressing cancer cells were demonstrated using immunofluorescence microscopy and quantified by an inductively coupled plasma-mass spectrometry dosage of Gd(III). Such an agent should enable the in vivo detection by MRI of tumors expressing Gb3 receptors.

A real-time cell-binding assay reveals dynamic features of STxB-Gb3 cointernalization and STxB-mediated cargo delivery into cancer cells.

The interaction between the Shiga toxin B-subunit (STxB) and its globotriaosylceramide receptor (Gb3) has a high potential for being exploited for targeted cancer therapy. The primary goal of this study was to evaluate the capacity of STxB to carry small molecules and proteins as cargo into cells. For this purpose, an assay was designed to provide real-time information about the StxB-Gb3 interaction as well as the dynamics and mechanism of the internalization process. The assay revealed the ability to distinguish the process of binding to the cell surface from internalization and presented the importance of receptor and STxB clustering for internalization. The overall setup demonstrated that the binding mechanism is complex, and the concept of affinity is difficult to apply. Hence, time-resolved methods, providing detailed information about the interaction of STxB with cells, are critical for the optimization of intracellular delivery.

Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways.

Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be achieved by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real time imaging of three different endocytic ligands - folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single particle tracking of Gal3 or STxB-functionalized, QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs that bear a unique stoichiometry of endocytic ligands represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics.

Gastric Adenocarcinomas Express the Glycosphingolipid Gb3/CD77: Targeting of Gastric Cancer Cells with Shiga Toxin B-Subunit.

The B-subunit of the bacterial Shiga toxin (STxB), which is nontoxic and has low immunogenicity, can be used for tumor targeting of breast, colon, and pancreatic cancer. Here, we tested whether human gastric cancers, which are among the most aggressive tumor entities, express the cellular receptor of Shiga toxin, the glycosphingolipid globotriaosylceramide (Gb3/CD77). The majority of cases showed an extensive staining for Gb3 (36/50 cases, 72%), as evidenced on tissue sections of surgically resected specimen. Gb3 expression was detected independent of type (diffuse/intestinal), and was negatively correlated to increasing tumor-node-metastasis stages (P = 0.0385), as well as with markers for senescence. Gb3 expression in nondiseased gastric mucosa was restricted to chief and parietal cells at the bottom of the gastric glands, and was not elevated in endoscopic samples of gastritis (n = 10). Gb3 expression in established cell lines of gastric carcinoma was heterogeneous, with 6 of 10 lines being positive, evidenced by flow cytometry. STxB was taken up rapidly by live Gb3-positive gastric cancer cells, following the intracellular retrograde transport route, avoiding lysosomes and rapidly reaching the Golgi apparatus and the endoplasmic reticulum. Treatment of the Gb3-expressing gastric carcinoma cell line St3051 with STxB coupled to SN38, the active metabolite of the topoisomerase type I inhibitor irinotecan, resulted in >100-fold increased cytotoxicity, as compared with irinotecan alone. No cytotoxicity was observed on gastric cancer cell lines lacking Gb3 expression, demonstrating receptor specificity of the STxB-SN38 compound. Thus, STxB is a highly specific transport vehicle for cytotoxic agents in gastric carcinoma. Mol Cancer Ther; 15(5); 1008-17. ©2016 AACR.

Targeted Shiga toxin–drug conjugates prepared via Cu-free click chemistry

The main drawback of the anticancer chemotherapy consists in the lack of drug selectivity causing severe side effects. The targeted drug delivery appears to be a very promising strategy for controlling the biodistribution of the cytotoxic agent only on malignant tissues by linking it to tumor-targeting moiety. Here we exploit the natural characteristics of Shiga toxin B sub-unit (STxB) as targeting carrier on Gb3-positive cancer cells. Two cytotoxic conjugates STxB–doxorubicin (STxB–Doxo) and STxB–monomethyl auristatin F (STxB–MMAF) were synthesised using copper-free ‘click’ chemistry. Both conjugates were obtained in very high yield and demonstrated strong tumor inhibition activity in a nanomolar range on Gb3-positive cells.

Influence of Gb3 glycosphingolipids differing in their fatty acid chain on the phase behaviour of solid supported membranes: chemical syntheses and impact of Shiga toxin binding

The Shiga toxin B subunit (STxB), which is involved in cell membrane attachment and trafficking of Shiga holotoxin, binds specifically to the glycosphingolipid Gb3. In biological membranes, Gb3 glycosphingolipids differ in their fatty acid composition and there is strong evidence that the fatty acid alters the binding behaviour of STxB as well as the intracellular routing of the Shiga toxin/Gb3 complex. To analyse the binding of STxB to different Gb3s, we chemically synthesized saturated, unsaturated, α-hydroxylated Gb3s and a combination thereof, all based on a C24-fatty acid chain starting from monosaccharide building blocks, sphingosine and the respective fatty acids. These chemically well-defined Gb3s were inserted into solid supported phase-separated lipid bilayers composed of DOPC/sphingomyelin/cholesterol as a simple mimetic of the outer leaflet of animal cell membranes. By fluorescence- and atomic force microscopy the phase behaviour of the bilayer as well as the lateral organization of bound STxB were analysed. The fatty acid of Gb3 significantly alters the ratio between the ordered and disordered phase and induces a third intermediate phase in the presence of unsaturated Gb3. The lateral organization of STxB on the membranes varies significantly. While STxB attached to membranes with Gb3s with saturated fatty acids forms protein clusters, it is more homogeneously bound to membranes containing unsaturated Gb3s. Large interphase lipid redistribution is observed for α-hydroxylated Gb3 doped membranes. Our results clearly demonstrate that the fatty acid of Gb3 strongly influences the lateral organization of STxB on the membrane and impacts the overall membrane organization of phase-separated lipid membranes.

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl(2)). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

The effects of the carboxyl-terminus amino acids of the Shiga toxin B-subunit on retrograde transport

The Shiga toxin B-subunit (STxB), from the enteric pathogen, Shigella dysenteriae, is responsible for the attachment of its receptor, globotriaosylceramide (Gb3), and navigates the retrograde pathway from the plasma membrane to the endoplasmic reticulum (ER). In this study, in order to demonstrate the role of carboxyl-terminus (C-terminus/al) amino acids of the B-fragment on the retrograde transport speed and the retrograde transport pathway, STxB was modified by site-directed mutagenesis and by the addition of an amino acid tail. The results showed that when the C-terminal amino acid, arginine [Arg (R)], was mutated to serine [Ser (S)], the speed of the B-fragment transportation into the ER at 37 ˚C was slower. When an acidic amino acid tail 'glutamine (Glu)-Ser' (ES) was added to the C-terminal amino acid 'R', the B-fragment transporting speed slowed down and remained in the Golgi apparatus. Further experiments showed that the effects induced by mutations of the amino acid tail resulted in STxB-EEEES ≥-EEES>-EES>-ES, demonstrating that the retardation effect on the tail was increased and the length of the acidic amino acid was augmented. The effect was possibly produced by an acidic amino acid tail, not only by the amino acid 'E'. The significant inhibitory effect on the speed of B-fragment retrograde transport was observed only when the mutations of the acidic amino acid tail were linked near to the C-terminus. These results may provide important insights for the study of transport mechanisms and for the development of STxB serial proteins as vectors for drug delivery.

Tumor-Specific Targeting of Pancreatic Cancer with Shiga Toxin B-Subunit

Pancreatic carcinoma is one of the most aggressive tumor entities, and standard chemotherapy provides only modest benefit. Therefore, specific targeting of pancreatic cancer for early diagnosis and therapeutic intervention is of great interest. We have previously shown that the cellular receptor for Shiga toxin B (STxB), the glycosphingolipid globotriaosylceramide (Gb(3) or CD77) is strongly increased in colorectal adenocarcinoma and their metastases. Here, we report an upregulation of Gb(3) in pancreatic adenocarcinoma (21 of 27 cases) as compared with matched normal tissue (n = 27). The mean expression was highly significantly increased from 30 ± 16 ng Gb(3)/mg tissue in normal pancreas to 61 ± 41 ng Gb(3)/mg tissue (mean ± SD, P = 0.0006), as evidenced by thin layer chromatography. Upregulation of Gb(3) levels did not depend on tumor stage or grading and showed no correlation with clinical outcome. Tumor cells and endothelial cells were identified as the source of increased Gb(3) expression by immunocytochemistry. Pancreatic cancer cell lines showed rapid intracellular uptake of STxB to the Golgi apparatus, following the retrograde pathway. The therapeutic application of STxB was tested by specific delivery of covalently coupled SN38, an active metabolite of the topoisomerase I inhibitor irinotecan. The cytotoxic effect of the STxB-SN38 compound in pancreatic cancer cell lines was increased more than 100-fold compared with irinotecan. Moreover, this effect was effectively blocked by competing incubation with nonlabeled STxB, showing the specificity of the targeting. Thus, STxB constitutes a promising new tool for specific targeting of pancreatic cancer.

Chemistry-based protein modification strategy for endocytic pathway analysis

The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large-scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means. Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor-made chemical tools that are then applied to the step-by-step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B-subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates. Our vectorial concept proposes a new chemical approach for traffic-based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.

Biodistribution and Tumor Targeting of Indium and Iodine-labeled Shiga Toxin B-Subunit

Strategies that aim at targeting radionuclides to human cancers have made important advances in recent years, and the need for improved delivery systems represents one of the current challenges in nuclear oncology. In this report, we have used the non-toxic B-subunit of Shiga toxin as a delivery tool. The synthesis of a maleimide-DOTA bifunctional chelating agent and a method for site-specific labeling a B-subunit variant with radioactive metals such as 111indium were developed. Biodistribution studies in mice revealed the accumulation of [111In]STxB in kidney and lung. Quantitatively and qualitatively similar results were obtained with iodinated STxB. The distribution half-life of [125I]STxB was 0.31 hours, and its elimination half-life was 9.54 hours, leading to a mean residence time of 12.8 hours, which is surprisingly high for such a small molecule. In a mouse model of spontaneous adenocarcinomatosis, a significant accumulation of [ In]STxB was observed in tumor tissue. Autoradiography of the excised digestive tube showed the accumulation of the vector in distinct tumor nodules. In conclusion, our study documents the potential use of STxB as a vector for tumor delivery of radionuclides. The relevance of these data with respect to the human situation, particularly kidney accumulation of [ In]STxB, is discussed.

EHD3 regulates early-endosome-to-Golgi transport and preserves Golgi morphology

Depletion of EHD3 affects sorting in endosomes by altering the kinetics and route of receptor recycling to the plasma membrane. Here we demonstrate that siRNA knockdown of EHD3, or its interaction partner rabenosyn-5, causes redistribution of sorting nexin 1 (SNX1) to enlarged early endosomes and disrupts transport of internalized Shiga toxin B subunit (STxB) to the Golgi. Moreover, under these conditions, Golgi morphology appears as a series of highly dispersed and fragmented stacks that maintain characteristics of cis-, medial- and trans-Golgi membranes. Although Arf1 still assembled onto these dispersed Golgi membranes, the level of AP-1 gamma-adaptin recruited to the Golgi was diminished. Whereas VSV-G-secretion from the dispersed Golgi remained largely unaffected, the distribution of mannose 6-phosphate receptor (M6PR) was altered: it remained in peripheral endosomes and did not return to the Golgi. Cathepsin D, a hydrolase that is normally transported to lysosomes via an M6PR-dependent pathway, remained trapped at the Golgi. Our findings support a role for EHD3 in regulating endosome-to-Golgi transport, and as a consequence, lysosomal biosynthetic, but not secretory, transport pathways are also affected. These data also suggest that impaired endosome-to-Golgi transport and the resulting lack of recruitment of AP-1 gamma-adaptin to Golgi membranes affect Golgi morphology.

Intracellular trafficking of Shiga‐toxin‐B‐subunit‐functionalized spherulites

Spherulites are multi-lamellar lipidic vesicles that can encapsulate biomolecules and may be used as carriers for drug delivery. STxB (Shiga toxin B-subunit) is known to bind the glycosphingolipid Gb3 (globotriaosyl ceramide), which is overexpressed by various human tumours. After Gb3 binding, the toxin enters the cytoplasm via the retrograde route, bypassing the degrading environment of the late endosomes/lysosomes. STxB is non-toxic and has been identified as a promising tool for drug delivery. So far, applications have relied on direct coupling with therapeutic agents. In the present study, we have investigated the functionalization of spherulites by STxB and the intracellular trafficking of these structures. We demonstrate that STxB-spherulites (ST x B-functionalized spherulites) are internalized into HeLa cells in a receptor-dependent manner. The intracellular distribution was studied by confocal microscopy for lipids, ligand and content. We observed an early separation between spherulites and STxB, leading to a late endosomal/lysosomal localization of lipids and content, whereas STxB remained partially at the plasma membrane. Although recognition of Gb3 is the cause of their specific adhesion to cell membranes, STxB-spherulites do not follow the retrograde transport route. Our results strongly suggest that STxB-spherulites are, at least in part, disrupted at the plasma membrane, leading to lipid and content targeting to the classical endocytic pathway. We discuss how these findings influence the development of innovative delivery strategies.

Specific adsorption of functionalized colloids at the surface of living cells: A quantitative kinetic analysis of the receptor-mediated binding

This paper presents a statistical experimental study of the adsorption of colloids onto the plasma membrane of living cells mediated by specific ligand-receptor interactions. The colloids consist of lipid multilamellar liposomes (spherulites) functionalized by Shiga toxin B-subunit (STxB), while cells are cervix carcinoma epithelial cells expressing the Shiga toxin receptor, the glycolipid globotriaosyl ceramide (Gb3). The specificity of the colloid adsorption is demonstrated using both confocal microscopy and flow cytometry, while a thorough cytometry study on living cells allows characterizing the kinetics of this specific adsorption. The final number of bound colloids and the characteristic adsorption time are shown to depend on bulk concentration, as expected for a thermodynamic equilibrium. However, the colloids appear to be irreversibly attached to the membrane. We interpret this apparent irreversibility as the result of a progressive recruitment of receptors. The methodology used here, whereby microscopic mechanisms are deduced from direct quantitative measurements on living cells, might allow the optimization of drug delivery systems or the quantification of virus infectivity.

Shiga toxin B-subunit sequential binding to its natural receptor in lipid membranes

Shiga toxin B-subunit (STxB), a protein involved in the cell-binding and intracellular trafficking of Shiga holotoxin, binds to a specific glycolipid, the globotriaosyl ceramide (Gb 3). Tryptophan residues of STxB, located at the protein–membrane interface, allow one to study its interaction with model membranes by means of spectroscopic methods with no need for chemical derivatisation with a fluorophore. The protein emits maximally around 346 nm and a blue shift of about 8 nm, as well as the occurrence of changes in the emission fluorescence intensity spectra, is indicative of insertion and partition into the membrane. However, the interaction seems to take place without pentamer dissociation. Acrylamide quenching experiments confirm tryptophan residues become less exposed to solvent when in the presence of vesicles, and the use of lipophilic probes suggests that they are located in a shallow position near the water/membrane interface. Fluorescence intensity and lifetime measurements upon STxB titration with Gb 3-containing vesicles suggest a complex STxB/Gb 3 docking mechanism involving static quenching in the later stages. Based on our observations, a model of the protein–membrane interaction is proposed and the STxB membrane partition and binding constants were calculated.

Shiga Toxin Facilitates Its Retrograde Transport by Modifying Microtubule Dynamics

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.

Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N −, 12C 15N −, 31P −) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N − from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

Characterization of the non-native trifluoroethanol-induced intermediate conformational state of the Shiga toxin B-subunit

The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.