scholarly journals Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

2021 ◽  
Author(s):  
Giuseppe Sicoli ◽  
Albert Konijnenberg ◽  
Jeremy Guerin ◽  
Steve Hessmann ◽  
Elise Del Nero ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Protein Transport ◽  
Integrative Approach ◽  
External Energy ◽  
Paramagnetic Resonance ◽  
Protein Insertion ◽  
Beta Barrel ◽  
Beta Hairpin

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane beta barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TspB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic beta hairpin immediately preceding the first barrel strand can insert into the beta barrel. We propose that these motions enlarge the channel and may hoist the substrate into it for secretion. An anchor region at the interface of the beta barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these proteins mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

scholarly journals ATP-induced asymmetric pre-protein folding as a driver of protein translocation through the Sec machinery

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Robin A Corey ◽  
Zainab Ahdash ◽  
Anokhi Shah ◽  
Euan Pyle ◽  
William J Allen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Transport ◽  
Protein Translocation ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Protein A ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Exchange Mass Spectrometry ◽  
Directional Diffusion ◽  
Protein Transporters

Transport of proteins across membranes is a fundamental process, achieved in every cell by the ‘Sec’ translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

scholarly journals The dynamic action of SecA during the initiation of protein translocation

2013 ◽  
Vol 449 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Vicki A. M. Gold ◽  
Sarah Whitehouse ◽  
Alice Robson ◽  
Ian Collinson
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Protein Transport ◽  
Protein Translocation ◽  
Cross Linking ◽  
High Affinity ◽  
Binding Domains ◽  
Alternative Position ◽  
Opening And Closing

The motor ATPase SecA drives protein secretion through the bacterial Sec complex. The PPXD (pre-protein cross-linking domain) of the enzyme has been observed in different positions, effectively opening and closing a clamp for the polypeptide substrate. We set out to explore the implicated dynamic role of the PPXD in protein translocation by examining the effects of its immobilization, either in the position occupied in SecA alone with the clamp held open or when in complex with SecYEG with the clamp closed. We show that the conformational change from the former to the latter is necessary for high-affinity association with SecYEG and a corresponding activation of ATPase activity, presumably due to the PPXD contacting the NBDs (nucleotide-binding domains). In either state, the immobilization prevents pre-protein transport. However, when the PPXD was attached to an alternative position in the associated SecYEG complex, with the clamp closed, the transport capability was preserved. Therefore large-scale conformational changes of this domain are required for the initiation process, but not for translocation itself. The results allow us to refine a model for protein translocation, in which the mobility of the PPXD facilitates the transfer of pre-protein from SecA to SecYEG.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Nitroxyl Modified Tobacco Mosaic Virus as a Metal-Free High-Relaxivity MRI and EPR Active Superoxide Sensor

2018 ◽  
Author(s):  
Madushani Dharmarwardana ◽  
André F. Martins ◽  
Zhuo Chen ◽  
Philip M. Palacios ◽  
Chance M. Nowak ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Single Molecule ◽  
Biological Fluids ◽  
Cellular Respiration ◽  
Organic Radical ◽  
Paramagnetic Resonance ◽  
Metal Free ◽  
Disease States

Superoxide overproduction is known to occur in multiple disease states requiring critical care yet non-invasive detection of superoxide in deep tissue remains a challenge. Herein, we report a metal-free magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR) active contrast agent prepared by “click conjugating” paramagnetic organic radical contrast agents (ORCAs) to the surface of tobacco mosaic virus (TMV). While ORCAs are known to be reduced <i>in vivo</i> to an MRI/EPR silent state, their oxidation is facilitated specifically by reactive oxygen species—in particular superoxide—and are largely unaffected by peroxides and molecular oxygen. Unfortunately, single molecule ORCAs typically offer weak MRI contrast. In contrast, our data confirm that the macromolecular ORCA-TMV conjugates show marked enhancement for <i>T<sub>1</sub></i> contrast at low field (<3.0 T), and <i>T<sub>2</sub></i> contrast at high field (9.4 T). Additionally, we demonstrated that the unique topology of TMV allows for “quenchless fluorescent” bimodal probe for concurrent fluorescence and MRI/EPR imaging, which was made possible by exploiting the unique inner and outer surface of the TMV nanoparticle. <a>Finally, we show TMV-ORCAs do not respond to normal cellular respiration, minimizing the likelihood for background, yet still respond to enzymatically produced superoxide in complicated biological fluids like serum.</a>

ДИНИТРОЗИЛЬНЫЕ КОМПЛЕКСЫ ЖЕЛЕЗА C ТИОЛСОДЕРЖАЩИМИ ЛИГАНДАМИ ПРЕДСТАВЛЕНЫ В ЖИВЫХ ОРГАНИЗМАХ В ОСНОВНОМ ИХ БИЯДЕРНОЙ ФОРМОЙ

2020 ◽  
Vol 65 (6) ◽  
pp. 1142-1153
Author(s):  
В.Д. Микоян ◽  
◽  
Е.Н. Бургова ◽  
Р.Р. Бородулин ◽  
А.Ф. Ванин ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Sodium Nitrite ◽  
Iron Complexes ◽  
Paramagnetic Resonance ◽  
First Case ◽  
Dinitrosyl Iron ◽  
Electron Paramagnetic ◽  
Dinitrosyl Complexes

The number of mononitrosyl iron complexes with diethyldithiocarbamate, formed in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite or the vasodilating drug Isoket® was assessed by electron paramagnetic resonance (EPR). The number of the said complexes, in contrast to the complexes, formed after nitrite or Isoket administration, the level of which sharply increased after treatment of liver preparations with a strong reducing agent - dithionite, did not change in the presence of dithionite. It was concluded that, in the first case, EPR-detectable mononitrosyl iron complexes with diethyldithiocarbamate in the absence and presence of dithionite appeared as a result of the reaction of NO formed from nitrite with Fe2+-dieth- yldithiocarbamate and Fe3+-diethyldithiocarbamate complexes, respectively. In the second case, mononitrosyl iron complexes with diethyldithiocarbamate appeared as a result of the transition of iron-mononitosyl fragments from ready-made iron-dinitrosyl groups of binuclear dinitrosyl complexes, which is three to four times higher than the content of the mononuclear form of these complexes in the tissue...

Naphthoquinones and derivatives for chemotherapy: perspectives and limitations of their anti-trypanosomatids activities

2020 ◽  
Vol 26 ◽  
Author(s):  
Luíza Dantas-Pereira ◽  
Edézio F. Cunha-Junior ◽  
Valter V. Andrade-Neto ◽  
John F. Bower ◽  
Guilherme A. M. Jardim ◽  
...  
Keyword(s):  
Large Scale ◽  
Neglected Tropical Diseases ◽  
Folk Medicine ◽  
Leishmania Species ◽  
Parasitic Infections ◽  
Mechanistic Analysis ◽  
Leishmania Spp ◽  
Nanomolar Range

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

Implantable neural interfaces

2018 ◽  
Author(s):  
Stefano Vassanelli
Keyword(s):  
Brain Function ◽  
Large Scale ◽  
Ionic Channels ◽  
Patch Clamp Technique ◽  
Advantages And Disadvantages ◽  
Optogenetic Stimulation ◽  
Physical Interfaces ◽  
The Brain

Establishing direct communication with the brain through physical interfaces is a fundamental strategy to investigate brain function. Starting with the patch-clamp technique in the seventies, neuroscience has moved from detailed characterization of ionic channels to the analysis of single neurons and, more recently, microcircuits in brain neuronal networks. Development of new biohybrid probes with electrodes for recording and stimulating neurons in the living animal is a natural consequence of this trend. The recent introduction of optogenetic stimulation and advanced high-resolution large-scale electrical recording approaches demonstrates this need. Brain implants for real-time neurophysiology are also opening new avenues for neuroprosthetics to restore brain function after injury or in neurological disorders. This chapter provides an overview on existing and emergent neurophysiology technologies with particular focus on those intended to interface neuronal microcircuits in vivo. Chemical, electrical, and optogenetic-based interfaces are presented, with an analysis of advantages and disadvantages of the different technical approaches.

What Has Direct Cortical and Subcortical Electrostimulation Taught Us about Neurolinguistics?

2019 ◽  
pp. 185-211
Author(s):  
Hugues Duffau
Keyword(s):  
White Matter ◽  
Large Scale ◽  
Functional Neuroimaging ◽  
Great Accuracy ◽  
Subcortical White Matter ◽  
Cerebral Lesions ◽  
Physiological Basis ◽  
Postoperative Mri ◽  
The Brain

Investigating the neural and physiological basis of language is one of the most important challenges in neurosciences. Direct electrical stimulation (DES), usually performed in awake patients during surgery for cerebral lesions, is a reliable tool for detecting both cortical and subcortical (white matter and deep grey nuclei) regions crucial for cognitive functions, especially language. DES transiently interacts locally with a small cortical or axonal site, but also nonlocally, as the focal perturbation will disrupt the entire subnetwork sustaining a given function. Thus, in contrast to functional neuroimaging, DES represents a unique opportunity to identify with great accuracy and reproducibility, in vivo in humans, the structures that are actually indispensable to the function, by inducing a transient virtual lesion based on the inhibition of a subcircuit lasting a few seconds. Currently, this is the sole technique that is able to directly investigate the functional role of white matter tracts in humans. Thus, combining transient disturbances elicited by DES with the anatomical data provided by pre- and postoperative MRI enables to achieve reliable anatomo-functional correlations, supporting a network organization of the brain, and leading to the reappraisal of models of language representation. Finally, combining serial peri-operative functional neuroimaging and online intraoperative DES allows the study of mechanisms underlying neuroplasticity. This chapter critically reviews the basic principles of DES, its advantages and limitations, and what DES can reveal about the neural foundations of language, that is, the large-scale distribution of language areas in the brain, their connectivity, and their ability to reorganize.

Sign in / Sign up

Export Citation Format

Share Document