Membrane translocation assay based on proteolytic cleavage: Application to diphtheria toxin T domain

Rodnin, Mykola V.; Ladokhin, Alexey S.

doi:10.1016/j.bbamem.2014.09.013

Cited by 6 publications

(14 citation statements)

References 22 publications

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“…Although membrane leakage is a very sensitive way of detecting protein-membrane interactions, our previous studies with various mutants indicate that it provides a less specific measure of physiological activity (26), especially when compared to the N-terminus translocation assay (48). Here, we applied this protease-based method, which we previously developed to study T domain activity, to examine the translocational activity of both proteins as a function of pH.…”

Section: Resultsmentioning

confidence: 99%

“…The N-terminus translocation assay was performed using thrombin-loaded large unilamellar vesicles (LUVs) made of a 3:1 molar ratio of POPC and POPG, as described previously (48). The recombinantly expressed T domain in pET15b plasmid contains an N-terminal His-tag with thrombin cleavage-site treatment.…”

Section: N-terminus Translocation Assaymentioning

confidence: 99%

“…After 1 h incubation at room temperature, 10 mL of 5Â SDS sample buffer was added, samples were boiled for 5 min and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) in 4%-15% Tris-HCl gels. Other details, including control experiments demonstrating that cleavage occurs inside the vesicles and that thrombin is active at pH R5.8, are described in (48).…”

Section: N-terminus Translocation Assaymentioning

confidence: 99%

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The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch

Rodnin

Gross

et al. 2016

Biophysical Journal

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Protein-side-chain protonation, coupled to conformational rearrangements, is one way of regulating physiological function caused by changes in protein environment. Specifically, protonation of histidine residues has been implicated in pH-dependent conformational switching in several systems, including the diphtheria toxin translocation (T) domain, which is responsible for the toxin's cellular entry via the endosomal pathway. Our previous studies a) identified protonation of H257 as a major component of the T domain's conformational switch and b) suggested the possibility of a neighboring H223 acting as a modulator, affecting the protonation of H257 and preventing premature conformational changes outside the endosome. To verify this "safety-latch" hypothesis, we report here the pH-dependent folding and membrane interactions of the T domain of the wild-type and that of the H223Q mutant, which lacks the latch. Thermal unfolding of the T domain, measured by circular dichroism, revealed that the reduction in the transition temperature for helical unfolding for an H223Q mutant starts at less acidic conditions (pH <7.5) relative to the wild-type protein (pH <6.5). Hydrogen-deuterium-exchange mass spectrometry demonstrates that the H223Q replacement results in a loss of stability of the amphipathic helices TH1-3 and the hydrophobic core helix TH8 at pH 6.5. That this destabilization occurs in solution correlates well with the pH-range shift for the onset of the membrane permeabilization and translocation activity of the T domain, confirming our initial hypothesis that H223 protonation guards against early refolding. Taken together, these results demonstrate that histidine protonation can fine-tune pH-dependent switching in physiologically relevant systems.

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Section: Resultsmentioning

confidence: 99%

Section: N-terminus Translocation Assaymentioning

confidence: 99%

Section: N-terminus Translocation Assaymentioning

confidence: 99%

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The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch

Rodnin

Gross

et al. 2016

Biophysical Journal

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“…This view is strongly supported by the high WT-like activity of OCS-blocking mutants H322Q, H323Q, and H372Q observed in both (a) N-terminus translocation in mutants of the T-domain relative to N-terminus translocation of the WT protein at pH 5.8. The assay is based on proteolytic cleavage of N-terminal segment preloaded into lipid vesicles followed by SDS-PAGE [49]. The data are normalized to the WT and are plotted against previously published OCS activity [38].…”

Section: Ocs: Critical Intermediate or Byproduct Of Translocationmentioning

confidence: 99%

“…In contrast, Pathway 1 indicates that the OCS is formed after the translocation, and passageway through the bilayer is formed via an unknown, possibly transient conformation. In order to distinguish between the two pathways, we will use new and published data to compare how various mutations affect the following measures of T-domain activity: (a) conductance in planar bilayers (i.e., formation of the OCS) [37,38,[45][46][47][48], (b) N-terminus translocation in vesicles [37,49], and ultimately (c) cell death assay based on inhibition of protein synthesis in vivo [37,46,48,50]. The starting structure on top corresponds to the crystal structure of the toxin at neutral pH [42], and consists of the C-domain (red), T-domain (helices color-coded according to OCS topology), and R-domain (green).…”

Section: Comparing the Two Translocation Pathwaysmentioning

confidence: 99%

Cellular Entry of Diphtheria Toxin Does Not Require Formation of the Open-Channel State by its Translocation Domain

Rodnin¹,

Vargas-Uribe²,

Ladokhin³

2018

Biophysical Journal

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Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation-the Open-Channel State (OCS)-as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo.

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Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

Kyrychenko,

Ladokhin

2023

The Chemical Record

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Fluorescence spectroscopy provides numerous methodological tools for structural and functional studies of biological macromolecules and their complexes. All fluorescence‐based approaches require either existence of an intrinsic probe or an introduction of an extrinsic one. Moreover, studies of complex systems often require an additional introduction of a specific quencher molecule acting in combination with a fluorophore to provide structural or thermodynamic information. Here, we review the fundamentals and summarize the latest progress in applications of different classes of fluorescent probes and their specific quenchers, aimed at studies of protein folding and protein‐membrane interactions. Specifically, we discuss various environment‐sensitive dyes, FRET probes, probes for short‐distance measurements, and several probe‐quencher pairs for studies of membrane penetration of proteins and peptides. The goals of this review are: (a) to familiarize the readership with the general concept that complex biological systems often require both a probe and a quencher to decipher mechanistic details of functioning and (b) to provide example of the immediate applications of the described methods.

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Membrane translocation assay based on proteolytic cleavage: Application to diphtheria toxin T domain

Cited by 6 publications

References 22 publications

The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch

The pH-Dependent Trigger in Diphtheria Toxin T Domain Comes with a Safety Latch

Cellular Entry of Diphtheria Toxin Does Not Require Formation of the Open-Channel State by its Translocation Domain

Fluorescent Probes and Quenchers in Studies of Protein Folding and Protein‐Lipid Interactions

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