Performance evaluation of Hyperledger Fabric-enabled framework for pervasive peer-to-peer energy trading in smart Cyber–Physical Systems

The in-depth collaboration of Cyber–Physical Systems (CPSs) and smart grids constitute the novel paradigm of distributed energy trading, in which computation and process control are managed in an adaptive Peer-to-Peer (P2P) manner. To further strengthen this collaboration, Hyperledger Fabric (HF) can be prominently considered as a mean to implement next-generation secure and intelligent communication. However, implementing real-world applications on this platform may concern performance issues. For the constructive exploration of these issues, initially, we design a novel P2P energy trading framework for improving resource utilization and consequently addressing the impending electricity crisis challenge. Thenceforward, we evaluate the results based on the different system operational parameters for establishing a proof-of-concept. For determining performance bottlenecks and best-configuration, these results are investigated independently by using the Nectar Research Cloud, thereby sustaining scalability. The proposed evaluation approach will largely contribute to determining the system operational-level parameters of enterprise applications that will utilize the HF platform as their communication tool-support. In addition, a benchmark is presented based on the Hyperledger Caliper tool to facilitate application designers and developers in the form of selecting an appropriate implementation model across the two latest stable HF model versions. The illustrative CPS-enabled energy trading scenario corroborates the feasibility of the proposed framework to foster the development of HF-assisted smart P2P energy trading mechanisms

MTRAP and Semaphorin-7A interact via their TSR and Sema domains.

<p>(<b>A</b>) Expression of individual domains of MTRAP and Semaphorin-7A. Schematics of the MTRAP ligand and Semaphorin-7A receptor as they would appear in the membrane are shown on the left with individual domains labelled plus a signal peptide (black box). The entire ectodomain and subfragments containing the individual domains of MTRAP (left blot) and Semaphorin-7A (right blot) were resolved by SDS-PAGE under reducing conditions and detected by Western blot using Streptavidin-HRP. Predicted molecular weights (kDa) are indicated in brackets; MTRAP has an additional processed band at around 30 kDa corresponding to the size of the Cd4d3+4-tag (TAG = Cd4d3+4-Biotin). (<b>B</b>) The two MTRAP TSR domains presented in tandem but not individually, bind Semaphorin-7A using the AVEXIS assay. Biotinylated entire ectodomains and individual domains of MTRAP (left graph) and Semaphorin-7A (right graph) were used as baits in the AVEXIS assay against Semaphorin-7A and MTRAP preys, respectively. The different Semaphorin-7A domains show no binding using this technique. Bar graphs represent mean ± SEM, n = 3. (<b>C</b>) MTRAP TSR 1+2 bind Semaphorin-7A with similar kinetics as the entire ectodomain of MTRAP. Serial dilutions of purified Semaphorin-7A were injected over biotinylated TSR1+2 immobilised on a streptavidin-coated sensor chip until equilibrium was reached (upper inset). Reference-subtracted binding data were plotted as a binding curve and an equilibrium dissociation constant calculated as before. A <i>K</i>_D of 1.96±0.03 µM (mean ± SEM) was calculated from three independent experiments (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003031#ppat.1003031.s004" target="_blank">Table S1</a>). A representative experiment is shown. No binding was observed with TSR1 or TSR2 individually (lower inset). (<b>D</b>) The Sema domain binds MTRAP with similar kinetics as the entire ectodomain of Semaphorin-7a. Serial dilutions of purified MTRAP were injected over the biotinylated Semaphorin-7A ectodomain or each individual domains (Sema, PSI and Ig-like) immobilised on a streptavidin-coated sensor chip. Binding was observed with the Sema domain (top graph) with a <i>K</i>_D of 0.83±0.43 (mean ± SEM), calculated from two independent experiments. No binding was observed with the PSI or Ig domains individually (bottom graph).</p

The MTRAP-Semaphorin-7A interaction is not influenced by glycans.

<p>(<b>A</b>) PNGase F treatment of Semaphorin-7A. Biotinylated Semaphorin-7A was incubated with PNGase F for 10, 30 or 60 minutes. Enzyme-treated and untreated Semaphorin-7A were resolved by SDS-PAGE under reducing conditions and detected by Western blotting using Streptavidin-HRP. (<b>B</b>) Binding of MTRAP to PNGase F-treated Semaphorin-7A was indistinguishable from untreated Semaphorin-7A using the AVEXIS assay. MTRAP was used as the prey against Semaphorin-7A baits. (+) = positive control, (−) = negative control. Bar graphs show mean ± SEM, n = 3. (<b>C</b>) PNGase F treatment did not quantitatively influence MTRAP binding to Semaphorin-7A using SPR. Three concentrations of purified monomeric MTRAP were injected over flow cells immobilised with PNGase F-treated and untreated Semaphorin-7A. Dissociation rate constants (<i>k</i>_d) were calculated to be 0.063±0.00007 s⁻¹ for PNGase F treated, and 0.061±0.00006 s⁻¹ for untreated Semaphorin-7A, by fitting a first order dissociation model to the washout phase of the binding curves. Shown are the normalized, averaged values ± SEM, n = 3. (<b>D</b>) MTRAP does not interact with sulphated glycoconjugates. Purified monomeric Semaphorin-7A, chondroitin sulphate A, chondroitin sulphate C, dextran sulphate, heparin and heparan sulphate were injected at 1 mg/ml over MTRAP immobilised on a streptavidin-coated sensor chip.</p

Semaphorin-7A is an erythrocyte receptor for MTRAP.

<p>(<b>A</b>) Schematic diagram showing how TRAP-like ligands are thought to play a key bridging role between the parasite and the target cell that is to be invaded. In this case, the cytoplasmic region of MTRAP interacts with the parasite actin-myosin motor that provides the power necessary for invasion. The extracellular region of MTRAP interacts with an erythrocyte receptor thereby providing the necessary traction for forwards movement of the parasite, driving host cell invasion. (<b>B</b>) Purified monomeric and pentameric MTRAP bound human erythrocytes relative to a negative control (pentameric Cd200). Unbound, wash and eluted material was resolved under reducing conditions by SDS-PAGE and detected by Western blotting using an anti-His antibody. Predicted monomer molecular weights are indicated in brackets. The pentamers are expected to split into the constituent monomers upon reduction. (<b>C</b>) Systematic screening identifies Semaphorin-7A as an MTRAP receptor. MTRAP was screened against an erythrocyte receptor protein library using the AVEXIS assay, either as a prey against 40 erythrocyte baits (top panel) or as a bait against 36 erythrocyte preys (bottom panel). A single interaction with Semaphorin-7A (protein number 23) was identified in both bait–prey orientations. Bar graphs represent means ± SD, n = 3. (<b>D</b>) MTRAP and Semaphorin-7A directly interact. Serial dilutions of purified monomeric Semaphorin-7A were injected over MTRAP immobilised on a streptavidin-coated sensor chip until equilibrium had been achieved (inset). Reference-subtracted binding data were plotted as a binding curve and the equilibrium dissociation constant was calculated using non-linear regression fitting of a simple Langmuir binding isotherm to the data. A <i>K</i>_D of 1.18±0.40 µM (mean ± SEM) was calculated from three independent experiments (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003031#ppat.1003031.s004" target="_blank">Table S1</a>). A representative experiment is shown.</p

SpaP-SpaP interactions analyzed by <i>in vivo</i> photocrosslinking and sequence co-variation.

<p>(A) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>E</i>. <i>coli</i> BL21 (DE3) expressing SpaP_T15X^FLAG in the absence of all other T3SS components. The sample is shown with and without UV-irradiation to induce photocrosslinking of <i>p</i>Bpa to neighboring interaction partners. (B) Immunodetection of chromosome-encoded SpaP^FLAG on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing plasmid-encoded SpaP_T15X. (C) Immunodetection of SpaP^FLAG and the inner MS ring protein PrgK on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing indicated SpaP-<i>p</i>Bpa mutants separated by 2-dimensional blue native/SDS PAGE. Full 2D gels are only shown for SpaP^FLAG scanned in the 800 nm channel. The 2D gel showing SpaP_M187X^FLAG +UV has been re-probed with antibody for PrgK and scanned in the 700 nm channel. PrgK indicates the position of the assembled needle complex. An overlay of FLAG and PrgK signals is shown on the right. The relevant slice of the 700 nm image showing PrgK at 25 kDa and the overlay of both channels showing the needle complex-associated bands have been aligned to the corresponding 2D image. (D) Interaction map of SpaP. Lines indicate predicted interactions with a normalized coupling score > 0.8 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s003" target="_blank">S3 Table</a>) at positions with experimentally identified SpaP-SpaP crosslinks (at least from one side). Positions with experimentally observed SpaP-SpaP interactions are shown in black, target positions only predicted are shown in light blue. Grey shading indicates TM helices. Only positions within or in close proximity to TM helices are shown. Abbreviations: chr—chromosomal.</p

Isolation and stoichiometry analysis of the SpaPR subcomplex of the needle complex.

<p>(A) Elution profile of the purified SpaPR^FLAG complex run on a Superdex 200 10/300 GL column. The peaks corresponding to the SpaPR^FLAG complex and 3xFLAG peptide are indicated. (B) Coomassie-stained SDS PAGE gel of purified SpaPR^FLAG complex and of its FLAG-deficient control (left). Immunodetection of SpaP (green) and SpaR^FLAG (red) on Western blot from purified SpaPR^FLAG complex separated by SDS PAGE (right). (C) Traces of indicated detector signals from size exclusion chromatography—multi angle laser light scattering of purified SpaPR^FLAG complex (left). ASTRA-calculated mass profile of total components of peak of purified SpaPR^FLAG complex (polypeptides and detergent, middle). ASTRA-calculated mass profile polypeptide components of peak of purified SpaPR^FLAG complex (right). (D) Native mass spectrum of the SpaPR^STREP complex. Peak series corresponding to the SpaP:SpaR^STREP complex in a 5:1 ratio is marked in red, with the most abundant charge state (14+) indicated. The peak series marked in blue corresponds to the same SpaPR complex bound to a ligand with a mass of approximately 710 Da, indicative of an associated phospholipid. Note that the measured mass for SpaPR heterohexamer (157.882 kDa) is heavier than the theoretically calculated mass (157.280 kDa). Abbreviations: Coo: Coomassie stained, WB: Western blot, RI: refractive index, LS: light scattering.</p

Screen of protein-protein interactions of SpaP and SpaR by <i>in vivo</i> photocrosslinking.

<p>(A) Protter visualization of SpaP presenting predicted TM topology, positions analyzed by <i>in vivo</i> photocrosslinking (thick stroke), and identity of interactions (colored). (B) As in (A) but showing SpaR. (C) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing indicated plasmid-complemented SpaP-<i>p</i>Bpa mutants separated by SDS PAGE. <i>p</i>Bpa mutations are denoted as “X”. Each sample is shown with and without UV-irradiation to induce photocrosslinking of <i>p</i>Bpa to neighboring interaction partners. Since the running behavior of crosslinked proteins often deviates from the calculated mass due to incomplete unfolding and since membrane proteins like SpaP often show an aberrant running behavior, the position of a crosslink on a gel does not easily allow drawing direct conclusions on the size of the crosslinked adduct. Crosslinked proteins identified by mass spectrometry or Western blotting are indicated. Other highlighted interactions shown in A and B were based on comparable SDS PAGE band pattern. (D) As in (C) but showing SpaR complemented from a low-copy number plasmid expressing SpaPQR^FLAGS. (E) As in (C) but expression of SpaP-<i>p</i>Bpa mutants from their chromosomal location. (F) As in (D) but expression of SpaR-<i>p</i>Bpa mutants from their chromosomal location. Abbreviations: J—PrgJ, P—SpaP, Q—SpaQ, S—SpaS.</p

Interactions among the export apparatus components SpaP, SpaQ, SpaR, and SpaS.

<p>(A) Immunodetection of SpaR^FLAG on Western blots of SDS PAGE-separated crude membrane samples of Δ<i>spaPQRS S</i>. Typhimurium expressing indicated SpaP-<i>p</i>Bpa mutants from a pT10-<i>spaPQR</i>^FLAG<i>S</i> plasmid. (B) Immunodetection of SpaP^FLAG on Western blots of SDS PAGE-separated crude membrane samples of Δ<i>spaPQRS S</i>. Typhimurium expressing indicated SpaR-<i>p</i>Bpa mutants from a pT10-<i>spaP</i>^FLAG<i>QRS</i> plasmid. (C) Immunodetection of SpaS_N258A^FLAG on Western blots of SDS PAGE-separated crude membrane samples of <i>S</i>. Typhimurium expressing indicated plasmid-complemented SpaP-<i>p</i>Bpa mutants. (D) As in (C) but assessing the SpaP-SpaS interaction in absence of the inner ring protein PrgK. (E) Immunodetection of SpaP^FLAG on Western blots of SDS PAGE-separated crude membrane samples of <i>S</i>. Typhimurium expressing chromosome-encoded indicated SpaP-<i>p</i>Bpa mutants in the presence or absence of the inner ring protein PrgK. (F) As in (E) but showing SpaR_M209X^FLAG. (G) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>E</i>. <i>coli</i> BL21 (DE3) expressing indicated SpaP-<i>p</i>Bpa mutants together with SpaQRS to form the SpaPR complex. (H) As in (F) but expressing SpaP_V170XQR^FLAGS to reveal the SpaP-SpaR interaction in <i>E</i>. <i>coli</i>.</p

Models of SpaP, SpaR, SpaQ, SpaS, and PrgJ in the T3SS needle complex and its assembly.

<p>(A) Model of the central SpaP complex with surrounding export apparatus components SpaQ, SpaR, and SpaS, and direct connection to the inner rod formed by PrgJ. These results suggest that SpaP, SpaR, and PrgJ form the socket structure on the periplasmic side of the inner membrane patch of the base. (B) Model of a view of the membrane patch of the needle complex from the cytoplasmic side highlighting SpaP, SpaQ, SpaR, and SpaS. (C) Model of needle complex assembly. The unified Sct nomenclature [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.ref023" target="_blank">23</a>] is shown in parenthesis.</p

Visualization and characterization of the pore formed by SpaP and SpaR.

<p>(A) Six selected class averages (4, 23, 29, 36, 55, 82) of negative-stained isolated SpaPR complexes imaged by electron microscopy. The length of the scale bar represents 50 Å. The two class averages at the top represent the SpaP₅ complex. Arrowheads in the class averages in the middle and at the bottom represent the anticipated position of SpaR on the SpaP₅ ring. The complete picture of all class averages can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s009" target="_blank">S4 Fig</a>. (B) Fluorescent streptavidin detection of SDS PAGE-separated biotin maleimide-labeled proteins of whole cell lysates, cell culture supernatant, periplasmic fraction, or cytoplasmic fraction of <i>S</i>. Typhimurium Δ<i>prgHIJK</i>, <i>flhD</i>::<i>tet</i> moderately overexpressing indicated proteins from a medium copy number plasmid (pT12). (C) Blue native PAGE and immunodetection of a high molecular weight complex formed by EPEA-tagged SpaP alone.</p