The assembly dynamics of the cytolytic pore toxin ClyA

Benke, Stephan; Roderer, Daniel; Wunderlich, Bengt; Nettels, Daniel; Glockshuber, Rudi; Schuler, Benjamin

doi:10.1038/ncomms7198

Cited by 89 publications

(204 citation statements)

References 63 publications

(147 reference statements)

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“…4B) shows that FMN binds to FL RNC . Non-native folding states, like for example unfolded and molten globular apoflavodoxin, do not show a change in slope of the titration data and thus they do not bind FMN (50). The titration data of Fig.…”

Section: Resultsmentioning

confidence: 98%

“…The major fluorescence contribution to the binding curves reflects the decrease in free FMN due to FMN binding to apoflavodoxin. The minor fluorescence component to the binding curves tracks the increasing number of flavodoxin molecules, as freshly generated flavodoxin is slightly fluorescent (50). Thus, the FMN fluorescence trace obtained after titrating native apoflavodoxin with FMN is complex, is not simply biphasic, and needs to be analysed by using equations 1 to 4 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…3A and B), indicating that FMN is bound. Binding of FMN to native apoflavodoxin occurs primarily through a very specific combination and geometry of hydrogen bonds and aromatic interactions (25,50). Thus, FMN-containing FL P and -5 P must be natively folded.…”

Section: Resultsmentioning

confidence: 99%

“…To avoid covalent dimerization of purified A. vinelandii (apo)flavodoxin, the single cysteine at position 69 was substituted by an alanine (50). The C69A variant is similar to wild-type (apo)flavodoxin (49,50). An additional F44Y mutation was introduced (39), and this protein variant is referred to as F44Y (apo)flavodoxin.…”

Section: Methodsmentioning

confidence: 99%

“…FMN fluorescence of the apoflavodoxin samples was measured in stirred fluorescence cuvettes during five minutes, before and after manual addition of FMN at the same KPPi concentration as the corresponding protein solution. C69A apoflavodoxin samples were made in 100, 70, 40 and 10 mM KPPi and F44Y apoflavodoxin samples were prepared in 100, 90,80,70,60,50,40,30,20, and 10 mM KPPi, respectively. Excitation was at 450 nm and emission was monitored at 540 nm.…”

Section: Fmn Bindingmentioning

confidence: 99%

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The ribosome regulates flavodoxin folding

Houwman¹

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A Novel Immunomodulator Delivery Platform Based on Bacterial Biomimetic Vesicles for Enhanced Antitumor Immunity

Hua

Yang

et al. 2021

Advanced Materials

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T cell activation‐induced cell death (AICD) during tumor pathogenesis is a tumor immune escape process dependent on dendritic cells (DCs). Proper immune‐modulatory therapies effectively inhibit tumor‐specific CD8+ T cell exhaustion and enhance antitumor immune responses. Here, high‐pressure homogenization is utilized to drive immunomodulator IL10‐modified bacteria to extrude through the gap and self‐assemble into bacterial biomimetic vesicles exposing IL10 (IL10‐BBVs) on the surface with high efficiency. IL10‐BBVs efficiently target DCs in tumor‐draining lymph nodes and thus increase the interaction between IL10 on BBVs and IL10R on DCs to suppress AICD and mitigate CD8+ T cell exhaustion specific to tumor antigens. Two subcutaneous peripheral injections of IL10‐BBVs 1 week apart in tumor‐bearing mice effectively increase systemic and intratumoral proportions of CD8+ T cells to suppress tumor growth and metastasis. Tumor‐specific antigen E7 is enclosed into the periplasm of IL10‐BBVs (IL10‐E7‐BBVs) to realize concurrent actions of the immunomodulator IL10 and the tumor antigen human papillomavirus (HPV) 16E7 in lymph nodes, further enhancing the antitumor effects mediated by CD8+ T cells. The development of this modified BBV delivery platform will expand the application of bacterial membranes and provide novel immunotherapeutic strategies for tumor treatment.

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Rapid Microfluidic Dilution for Single‐Molecule Spectroscopy of Low‐Affinity Biomolecular Complexes

et al. 2017

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To enable the investigation of low-affinity biomolecular complexes with confocal single-molecule spectroscopy, we have developed amicrofluidic device that allows aconcentrated sample to be diluted by up to five orders of magnitude within milliseconds,atthe physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low-affinity protein complexes using single-molecule two-color and threecolor Fçrster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from lownanomolar up to 10 mm,t hus covering aw ide range of biomolecular interactions.T he design and precise fabrication of the devices ensure simple yet reliable operation and high reproducibility of the results. Single-moleculespectroscopyhasdevelopedintoapowerfulapproach for investigating biomolecular structure,d ynamics, and interactions,e specially in combination with Fçrster resonance energy transfer (FRET). [1] However,amajor challenge for studying the mechanisms of biomolecular interactions by single-molecule spectroscopy,s uch as FRET between two binding partners,isthat their affinities are often so low that they rapidly dissociate at the about 10 to 100 pm sample concentrations required for single-molecule detection. As ar esult, single-molecule studies with two fluorescently labeled interaction partners are often not possible at equilibrium. This limitation can be circumvented by forming the complex at high concentrations,r apidly diluting the sample to single-molecule concentrations,and monitoring its properties before dissociation. [2] However,there are currently no methods available that combine sufficiently large dilutions and short dead times to enable observation of such complexes before they dissociate.H erein, we demonstrate as olution to this problem using am icrofluidic device that enables the sample to be diluted more than 10 000-fold within milliseconds and allows the properties of the complex and its dissociation kinetics to be monitored by confocal singlemolecule spectroscopy.As uitable rapid dilution microfluidic device must meet several requirements.F irst, the dilution needs to be sufficiently rapid that even low-affinity complexes with high dissociation rates can be studied. Them inimum time for dilution in laminar flow is fundamentally limited by translational diffusion. Given the diffraction-limited size of the confocal observation volume and the diffusion coefficient of typical biomolecular samples,t he minimum dead time for a10000-to 100 000-fold dilution, for example,isinthe range of af ew milliseconds ( Figure S1 in the Supporting Information). Second, since not all applications require the same dilution factor,the device must provide dilutions that can be adjusted over aw ide range (about 1000-to 100 000-fold). Third, because high sample concentrations are required to form stable biomolecular complexes initially,t he device should have alow sample ...

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The assembly dynamics of the cytolytic pore toxin ClyA

Cited by 89 publications

References 63 publications

The ribosome regulates flavodoxin folding

The ribosome regulates flavodoxin folding

A Novel Immunomodulator Delivery Platform Based on Bacterial Biomimetic Vesicles for Enhanced Antitumor Immunity

Rapid Microfluidic Dilution for Single‐Molecule Spectroscopy of Low‐Affinity Biomolecular Complexes

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