Human observers can perceive the existence of a transparent surface from dynamic image deformation. They can also easily discriminate a transparent solid material such as plastic and glass from a transparent fluid one such as water and shampoo just by viewing them. However, the image information required for material discrimination of this sort is still unclear. A liquid changes its contour shape non-rigidly. We therefore examined whether additional properties of the contour of a deformation-defined region, which indicated contour non-rigidity, biased percepts of the region toward liquid materials. Our stimuli had a translating circular region wherein a natural texture image was deformed at the spatiotemporal deformation frequency that was optimal for the perception of a transparent layer. In Experiment 1, we dynamically deformed the contour of the circular region and found that large deformation of the contour biased the percept toward liquid. In Experiment 2, we manipulated the blurriness of the contour and observed that a strongly blurred contour biased percepts toward liquid. Taken together, the results suggest that a deforming region lacking a discrete contour biases percepts toward liquid. Recognizing materials is a complex but important task for the visual system. For example, by differentiating solid from liquid materials, humans can adaptively control their actions toward an object, such as how to grasp it . The experiments were conducted according to the principles laid down in the Helsinki Declaration. Written informed consent was obtained from all participants.Stimuli were presented on a 21-inch CRT monitor  with a resolution of 1024 × 768 pixels and a refresh rate of 60 Hz. A computer  controlled stimulus presentation and data collection with Psychopy v1.83 Peirce, , 2009.Stimuli contained a horizontally or vertically translating circular region wherein an image of a natural scene was non-rigidly deformed Figures . Ten natThe experiments were conducted in a dimly lit room. Each observer was tested individually, and sat at 60 cm from the CRT display. On each trial, a neutral gray blank field was initially presented for 1 s, followed by the stimulus image sequence. The sequence, consisting of 120 images, lasted for 2 s. After the disappearance of the image sequence, a neutral gray blank field was again presented. The observers were asked to carefully view the image sequence, and after its disappearance, rate perceived material category on a 5-point scale: “1” for a solid material, “5” for a fluid material, and intermediate values for intermediate impressions, with an explicit instruction that “3” meant the material category was vague. Each session consisted of 48 trials consisting of 4 (translation directions) × 6 (levels of contour deformation magnitudes) × 2 (repetitions); in each trial the background image was randomly selected from among the 10 images previously chosen from the image database. Each observer experienced four sessions, and so it took ~40 min to complete all four. The order of trials was pseudo-randomized across the sessions within an observer, and across observers.p < 0.05), we assessed whether the mean rating score for each level of contour deformation was statistically deviated from the value of “3” (the vague impression). The results showed that in all translation direction conditions, the rating scores were significantly greater than “3” when the magnitude of contour deformation was 0.90 deg . In all translation direction conditions except the leftward condition, the rating scores were significantly greater than “3” when the magnitude of contour deformation was 0.72 deg. In the downward condition, the rating scores were significantly greater than “3” when the magnitude of contour deformation was 0.54 deg. On the other hand, the rating scores were not significantly lower than “3” in all of the translation direction conditions.For each observer, the rating scores for the material impressions were averaged across repetitions. Mean rating scores across the observers are plotted in Figure F = 2.286, p = 0.104, partial η2 = 0.2]. The main effect was significant for the intercept . Multiple comparison tests . The results indicate that the downward translation biases the percept of deforming regions toward liquid.To check the statistical significance of the effect of translation direction on the rating scores, we assessed the slope and intercept of a linear function fitted to rating scores as a function of the magnitude of contour deformation Figure . The slots Ryan,  showed td′-value as a function of the magnitude of contour deformation by means of the procedure as described in the previous study  were available for each magnitude of contour deformation, we averaged them. Figure d′-values with 95% confidence intervals around the mean cumulative d′ for each level of contour deformation, collapsed across translation directions. We conducted a two-tailed one-sample t-test to check whether the cumulative d′ for each level of contour deformation was statistically different from zero. The cumulative d′ was not significantly deviated from 0 when the magnitude of contour deformation was 0.18 deg  while it was significantly higher than 0 when the magnitudes of contour deformation were 0.36 deg , 0.54 deg , 0.72 deg , and 0.90 deg . We also checked decision criteria c. Values above and below c lead to the response “signal is present” and “signal is absent,” respectively. We found that c  for yielding rating scores between 3 and 4 was included in the range of our stimulus manipulation, but not for cs between 1 and 2 (−0.93 ± 0.24), between 2 and 3 (−0.3 ± 0.29), and between 4 and 5 (1.233 ± 0.46). Figure d′-values. Clearly, there were a couple of participants who showed no effect of contour deformation. The individual difference may lead to the large confidence intervals for d′ and c, and may make the statistical analyses sensitive to outliers.To check to what extent the perceptual dimension of “liquidness” was affected by contour deformation, on the basis of signal detection theory  of the Gaussian blur filter that was applied to a circular region at six levels: 0, 0.06, 0.12, 0.18, 0.24, and 0.3 deg  × 2 (repetitions). Each observer received four sessions, and so it took ~40 min to complete all four.The procedure was also identical to that as used in Experiment 1 except for the following. Each session consisted of 48 trials consisting of 4 (translation directions) × 6 , we assessed whether the mean rating score for each level of contour deformation was statistically deviated from the value of “3” (the vague impression). The results showed that the rating scores were significantly larger than “3” when the SD was 0.24 deg in all translation direction conditions except the downward condition, and when the SD was 0.3 deg in the rightward and leftward conditions . Moreover, the rating scores was significantly lower than “3” when the SD was zero in all translation direction conditions except the downward condition.For each observer, the rating scores for the material impressions were averaged across repetitions. Mean rating scores across the observers are plotted in Figure F = 1.478, p = 0.2428, partial η2 = 0.14]. The main effect was not also significant for the intercept . Inconsistent with the results of Experiment 1, the results of this experiment showed no higher bias in the downward than other conditions for perceiving deforming regions toward liquid. At this stage it is unclear why there was the difference in the bias of the downward condition between the experiments. There are several possible explanations for the difference. First, the effect of translation directions on the bias toward a liquid impression may appear only when the contour of deforming regions is deformed. Second, because the observers had already participated in Experiment 1 and learned the statistics of the stimuli in the experiment, the natural bias for the downward translation may have been reduced in this experiment. Finally, the bias is possibly small and given that the number of repetitions at each blur was very low, the noise in the data might drown out the effect. It is necessary for future studies to closely address these possibilities.To check the statistical significance of the effect of translation direction on the rating scores, we assessed the slope and intercept of a linear function fitted to rating scores as a function of the magnitude of contour deformation Figure . For thed′-value as a function of the SD. Figure d′-value with 95% confidence intervals around the mean cumulative d′ for each level of contour deformation, collapsed across translation directions. We conducted a two-tailed one-sample t-test to check whether the cumulative d′ for each level of the SD of the Gaussian blur filter was statistically different from zero. The cumulative d′ was not significantly deviated from zero when the SD was 0.06 deg  and 0.12 deg . Meanwhile, the cumulative d′ was significantly higher than zero when the SD was 0.18 deg , 0.24 deg , and 0.30 deg . We also found that c  for determining the rating scores between 3 and 4 was included in the range of our stimulus manipulation, but not for cs between 1 and 2 (−0.34 ± 0.037), between 2 and 3 (−0.09 ± 0.093), and between 4 and 5 (0.537 ± 0.137). Figure d′-values. Similarly to Experiment 1  = 1.951, p > 0.8]. Hence, we would like to conclude that there was no essential difference in the rating values of the 0 deg conditions between the experiments. As shown by the analysis of c, no decision boundary for a solid material impression existed in the range of our stimulus manipulations. Thus, we suggest it is safe to conclude that our stimulus manipulation did not strongly contribute to the formation of a solid material impression.In this experiment, the rating scores in the 0 deg condition were significantly deviated from “3” while those in the 0 deg condition were not in Experiment 1, though both conditions involved identical stimulus. By using a two-tailed We examined how the non-rigidity of the contour of a deforming region biased the percept of the region toward liquid. In Experiment 1, we observed that the large contour deformation reliably biased the percept toward liquid. In Experiment 2, we showed that spatially blurring the contour of a deforming region biased the percept toward liquid.Our study successfully showed that the non-rigid deformation of the contour of a deforming region biased the percept toward liquid. In our stimuli, the pattern of image deformation within a deforming region rigidly moved along its trajectory in all conditions. Thus, the visual system possibly used the non-rigid deformation and/or the spatial blur of the contour of a deforming region to interpret the rigidly moving image deformation. The results are consistent with previous studies proposing that the information from the contour is used to perceptually determine the shape and/or material properties of the surface Knill, .A link between the perception of non-rigid contours and the perception of liquid was shown in the present study. Previous studies have reported several image features such as shape . There is a pending patent involving the reported research. There are no products in development or marketed products to declare. The pending patent does not alter the author's adherence to policies of Frontiers.


E.coli cis-isoprenyl transferase (UPPs). Small Angle X-ray Scattering (SAXS) analysis reveals the radius of gyration (Rg) of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax) of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD) analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.The Nogo-B receptor (NgBR) is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the  The Nogo-B receptor (NgBR) plays an essential role in angiogenesis during wound healing and embryonic development –4. In woRecently, we showed that NgBR is involved in breast cancer cell growth through modulating Ras signaling . NgBR prE.coli cis-IPtase. The similar radius of gyration (Rg), Porod volume, and Dmax indicate NgBR folds as a single globular domain in solution as the cis-IPtase does. However, this result conflicts with the previously proposed topology of NgBR at the plasma membrane which might partition this construct into three separate domains. This conflict raises a possibility of the presence of another topological orientation for NgBR.The structure of NgBR is currently unknown. Previous circular dichroism (CD) spectroscopy and NMR data showed that the cytosolic domain of NgBR is only partially folded and its extracellular region intrinsically disordered . It also6-SUMO tag. The vector was transformed into BL21(DE3) cells for recombinant protein expression. Cells were inoculated into LB media and grew to an optical density of 0.5. Cells were then induced with 0.1 mM isopropylthio-β-D-galactoside (IPTG) and grew at 15°C overnight. After harvest, cells were lysed using a French Press, and the supernatant was collected for purification. After the His6-SUMO tag was cut by yeast SUMO protease 1, the native NgBR(79–293) was separated from the tag by a second Ni2+ column. Finally, NgBR was purified by a size exclusion column  in 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol and 1 mM Tris(2-carboxyethyl)phosphine (TCEP).NgBR was essentially expressed and purified as previously described , 12. In g) was calculated using the Guinier approximation. Pair distribution function (P(r)) and the maximum particle dimension (Dmax) were estimated from the scattering data using the GNOM algorithm. An ab initio dummy atom model was generated using DAMMIN  BESTSEL . The secE.coli cis-IPtase undecaprenyl pyrophosphate synthase (UPPs) (NgBR was considered as a type I transmembrane protein at the plasma membrane . It conse (UPPs) . This lee (UPPs) . The fare (UPPs) . These re (UPPs) . Withoute (UPPs) . This ise (UPPs)  10]. Ho. HoE.colAdditional sequence similarity is present in NgBR transmembrane domain and some extracellular regions . The traE.coli. Over 50% of total recombinant NgBR present in the cell lysis was recovered in the supernatant fraction  that contains all conserved residues for NgBR to form a complete, UPPs-like structural fold and farnesyl binding pocket (NgBR(79–293)). The construct includes the entire cytosolic region, the transmembrane domain and some extracellular regions . The confraction . In contfraction . This infraction . The sizq range with a slight variation found at increasing q values  needs to fold into a single globular domain in order to form a structural fold that can have a farnesyl binding pocket similar to that of UPPs. However, if it adopts the currently proposed topology, this construct might not fold into a single globular domain, and might be partitioned into three separate regions. To determine the fold states of NgBR(79–293), Small Angle X-ray Scattering (SAXS) was performed with the purified protein. SAXS is a method that provides information on protein size, shape and fold states, and can distinguish between single-domain globularly-folded proteins and multi-domain proteins with flexible linkers and intrinsically disordered regions. Thus, SAXS analysis of NgBR should be able to reveal the fold states of NgBR in solution. To ensure monodispersity of the sample, NgBR(79–293) was subjected to synchrotron SAXS in-line with size-exclusion chromatography. Concentrated NgBR consisting of monomer, dimer, and tetrameric states were separated by size-exclusion chromatography followed by in-line X-ray scattering on the elution from the column . Only thq values . This inq values . Notably domains .g) value for NgBR is 18.2 Å which closely coincides with the Rg of UPPs (18.6 Å) (max) of NgBR is 61.0 Å similar to UPPs Dmax of 58.6 Å. Analysis of NgBR pair distribution function reveals a bell-shaped peak which is characteristic of a globularly folded protein  of 23.0 kD . The radius of gyration (R(18.6 Å) . The max protein . The Por 23.0 kD . This is 23.0 kD . AltogetTo further determine that NgBR(79–293) is folded, the secondary structure of this construct was probed by circular dichroism (CD) spectroscopy. The spectra reveal a strong broad negative band between 200 nm and 235 nm . The lowE.coli cis-IPtase. This construct is well folded without any evidence of structural disorder or characteristics of a multi-domain protein. However, our data appears to be in disagreement with the currently proposed topological orientation of NgBR which might separate this construct into three domains: cytosolic, transmembrane and extracellular. One possible explanation for this discrepancy is that NgBR can adopt another topological conformation and the construct NgBR(73–293) may represent a cytosolic region of a new, not-yet-identified topology.The high sequence similarity between NgBR and the farnesyl-binding protein cis-IPtase suggests that these two proteins may share a similar overall structural fold and farnesyl binding mode. However, the cytosolic region of NgBR itself is insufficient and unlikely to form a complete farnesyl binding pocket due to the lack of several conserved secondary structural elements. The recombinant construct of the cytosolic region is only partially folded and largely insoluble. However, we show that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region can significantly enhance the recombinant protein solubility. SAXS analysis of this protein construct demonstrates that it forms a single globular domain in solution with an overall size and shape similar to E.coli LacY where deficiency of phosphatidylethanolamine results in the inversion of its N-terminal six-transmembrane helical bundle and translocation of its seventh transmembrane domain to the extracellular space [NgBR has been proposed to adopt several topological conformations , 5, 10. ar space , 21. A sar space . With evS1 File(ZIP)Click here for additional data file.


Bacterial autotransporters comprise a C-terminal β-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated β-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter β-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the β-hairpin structure of the fifth extracellular loop of the β-barrel domain has a crucial role for passenger domain folding into a β-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with β-helical passenger domains. We propose that the autotransporter β-barrel domain is a folding vector that nucleates folding of the passenger domain. E. coli autotransporters Pet and EspP and propose that the β-barrel anchor acts as a vector to nucleate the folding of the passenger domain.Autotransporter passenger domains are presented on or released from the bacterial surface upon translocation through an outer membrane β-barrel anchor. Here the authors study the two  Although the different subtypes of type V autotransporters are distinguished by variations in their domain organization, oligomerization state, and structure, they are unified by a similar transmembrane topology1. Embedded in the outer membrane is a β-barrel domain that forms a secretion pore, which is needed for the translocation of the  passenger domain to the bacterial cell surface1.Type V autotransporter proteins (currently classified as types Va to Ve) are a distinct class of outer membrane proteins (OMPs) that share a common secretion pathway, which facilitates their navigation through the bacterial cell envelope2 that, in most cases, is folded or predicted to fold into an elongated β-helix6, although globular folds have also been reported7 and predicted to occur6. Once folded, passenger domains have key roles in pathogenesis; some passenger domains remain attached to the β-barrel to function as adhesins, whereas others are processed and released into the environment to function as toxins, or to mediate disruption of the host immune response11.The prototypical type Va  autotransporter consists of a 12-stranded C-terminal β-barrel domain and an N-terminal passenger domain14, as do periplasmic chaperones such as Skp and SurA18. Based on evidence from cross-linking studies that show autotransporters directly interact with BamA, the central component of the β-barrel assembly machinery20, it has been proposed that the passenger domain is translocated through a pore formed by the transient fusion of the autotransporter and BamA β-barrels during membrane insertion. BamA structures and molecular dynamics simulations show conformational flexibility along the seam that hydrogen bonds the first with the last β-strand of the BamA β-barrel22. However, it is unclear whether BamA can transiently form fusion pores with substrate OMPs.In a cellular context, key molecules catalyze classical autotransporter folding and membrane insertion as they do for other types of β-barrel proteins. The translocation and assembly module participates in autotransporter assembly24. It has been proposed that folding of the portion of the passenger domain that appears first at the cell surface into a stable scaffold nucleates processive folding of the more N-terminal segments of the passenger domain during translocation. In support of this hypothesis are chemical25 and mechanical26 denaturation studies showing that the core is the most stable part of the protein. Moreover, several lines of evidence suggest that stacking (π–π) interactions between buried aromatic residues in the stable core not only provide stability to this region, but also drive translocation and vectorial folding of the passenger domain27. Although collectively these results provide insight into how the passenger domain is translocated and folded into its functional form, many mechanistic details are lacking.Irrespective of the composition of the translocation pore, there is a general agreement that in classical autotransporters, the passenger domain is translocated in a C- to- N-terminal direction where the most C-terminal β-helical rungs fold into a stable, protease-resistant structure called the ‘stable core’Escherichia coli28 as a model protein, we present evidence that the fifth β-hairpin loop (L5), which connects β-strands 9 and 10 at the extracellular surface of the β-barrel domain, templates folding of the passenger domain. By monitoring the assembly of Pet variants carrying modifications in the L5 β-hairpin in live bacterial cells, we provide evidence that β-strand propensity of the loop, but not its specific amino acid sequence is required for folding of the passenger domain into a stable β-helix. Moreover, biophysical studies on refolded protein suggest that the nucleation of passenger domain folding can occur independently of the cellular machinery, and is entirely mediated by the β-barrel domain. Experimental analysis of a second classical autotransporter, EspP, and in silico sequence and structure comparison with other autotransporters lead us to suggest that L5-assisted folding of the passenger domain represents a conserved folding mechanism for classical autotransporter proteins.Here, using the classical Pet autotransporter, an enterotoxin from enteroaggregative E. coli has been crystallized30, thus providing a means to model the topology of the Pet β-barrel domain  subfamily of autotransporters, which are characterized by an autocatalytic processing of the passenger from the β-barrel between two conserved asparagine residues in the intra-barrel α-helical segment that connects both domains32  into distinct passenger and β-barrel fragments was in process by 5 min and largely completed by 10 min  had no effect on the stability of this domain, the authors also noted no effect on passenger domain translocation or cleavage.Truncation of a long β-hairpin loop (L4) that connects β-strands 7 and 8 at the extracellular surface of the β-barrel domain of BrkA, a classical autotransporter from ain Fig. , green aain Fig. , 7 and 8ain Fig. , and commin Fig. , suggestE. coli BW25113ΔompT36, a strain lacking the outer membrane protease OmpT. OmpT digests unfolded passenger domains on the bacterial surface, while natively folded passengers are resistant to OmpT degradation37. As expected, pulse-chase expression assays showed that the assembly of Pet in E. coli TOP10 ] only contained the 116 residues of the passenger domain immediately N-terminal to the L5-truncated β-barrel domain where the small passenger size (~ 13 kDa) would make it challenging to accurately pick up folding defects using pulse-chase assays29.To account for the disappearance of the ~ 106 kDa mature PetΔL5 passenger, pulse-chase expression of Pet and PetΔL5 was repeated in ompT visualized greater amounts of the 136 kDa and 106 kDa species  mutated to glycine, demonstrated a severe defect in passenger domain folding similar to that observed for PetΔL5  mutated to glycine, relative to that observed in wild type Pet  mutated to glycine, was similarly unaffected  in the first β-strand in L5 mediate folding of the Pet passenger domain.To identify the amino acid residues of L5 responsible for mediating passenger folding, Pet variants were created Fig.  and analΔL5 Fig. . As a sme K Fig. , + PK, wPet Fig. . PetL5Unted Fig. . TogetheL5VLI/N) or in which charged residues were swapped to the opposite charge  to disrupt potential salt bridges with the second β-strand  mutated to alanine, relative to that observed in wild-type Pet . To further validate whether disruption of the β-sheet structure perturbs passenger folding, we substituted all amino acids in the first β-strand in L5 for proline to create PetL5β1/P, eliminating potential hydrogen bonding partners required for the formation of a β-sheet  of the passenger located immediately N-terminal to the β-barrel domain  of 321 nm, whereas the spectrum of the PetΔL5Δ1-554 passenger appeared almost indistinguishable from that of the unfolded protein in guanidine hydrochloride, with a red-shifted λmax of 331 nm  shows L5 in a ‘closed’ conformation, folded into the pore of the β-barrel where it hydrogen bonds with several β-strands. In contrast, the crystal structure of the EspP β-barrel domain with the most C-terminal portion of the passenger domain still attached (PDB code 3SLJ) shows L5 in an ‘open’ conformation, projecting into the extracellular space. Here, the uncleaved passenger domain traverses the length of the pore, thereby physically blocking L5 from making extensive contact with the β-barrel. Instead, the second β-strand in L4 interacts with the first β-strand in L5, forming a four-stranded β-sheet , concerted and multistep process involving the entire protein, rather than vectorial54. By contrast, autotransporter assembly in vivo occurs rapidly (~ 10 min)18 and is in line with the typical doubling time of E. coli (~ 20 min). We conclude that the mechanistic differences between passenger folding with and without the β-barrel have transpired due to the inability to recapitulate in vitro the effects of the autotransporter β-barrel on passenger domain folding in the bacterial cell.L5-assisted folding of the passenger domain can explain the molecular basis for folding defective phenotypes observed in previous studies that investigated the in vitro folding of passengers in isolation from their cognate β-barrels. Studies on pertactin, a classical autotransporter from 25, it is also feasible that the β-barrel accelerates passenger folding through promotion of vectorial folding during translocation. This would allow the passenger C terminus to explore its conformational space before the N terminus has been translocated, thereby decreasing the probability of populating non-native structures. Such non-native structures likely cause slow in vitro refolding kinetics because they require extensive reorganization before the native conformation can be reached54. Vectorial folding would also ensure that the correct part of the passenger is able to interact with the first β-strand in L5 at the very beginning of the translocation process.In addition to actively augmenting folding, as proposed previouslyIn summary, we have discovered that the SPATE β-barrel domain is a folding vector that can act independently of the cellular machinery to promote folding of the passenger domain on a biologically relevant timescale and we provide evidence to show that the efficiency of this process is dependent on the L5 β-hairpin. The significance of our work is evident in the inability of the passenger domain to fold into its biologically active form in the absence of its interaction with L5, a phenotype that could be exploited for therapeutic purposes.−1 kanamycin and/or 100 μg mL−1 ampicillin, 0.2% (vol/vol) d-glucose, 0.02% (vol/vol) l-arabinose, or 0.5 mM isopropylthio-β-galactoside. Details of the E. coli strains  and plasmids used in this study are presented in Supplementary Table Bacteria were grown at 37 °C in Luria–Bertani broth and where necessary, the growth medium was supplemented with 30 μg mL34. pBADEspP, pBADEspPΔL5, and all pBADPet derivatives except PetL5β1/P were synthesized de novo by GenScript. To construct PetL5β1/P, megaprimer PCR was performed as described previously55. Briefly, the round 1 PCR was performed using 500 ng of template DNA (pBADPet) with 1 μg mL−1 each of primers MPL5β1Pro and HindIIIPetRv. The round 2 PCR was performed with 4 μg of megaprimer and 1 μg of primer SalIPetFw using 500 ng of template DNA (pBADPet). The resulting amplicon and target vector (pBADPet) were then digested with HindIII and SalI and ligated. To construct pETPetΔ1-554 and pETPetΔL5Δ1-554 the Pet β-barrel domain and the last 464 residues of the Pet passenger domain were amplified using 500 ng of template DNA (pBADPet and pBADPetΔL5) with 1 μg mL−1 each of primers NdeIPet464Fw and XhoIPetRv. The subsequent amplicons and target vector (pET-22b + ) were then digested with NdeI and XhoI, and ligated for an in frame C-terminal hexahistidine-tag fusion. All DNA modifications were confirmed by sequencing.pBADPet has been described previously56 was used to express protein as previously described34. In all cases, bacterial cultures were grown to an OD600 of ~ 0.6 in media containing 0.2% glucose to repress protein synthesis prior to harvesting . For pulse-chase expression of the pBADPet derivatives, the bacterial cells were resuspended in media containing 0.02% arabinose for five minutes (pulse) to induce Pet expression, collected, and then resuspended in media containing 0.2% glucose to “chase” the protein produced during the “pulse” phase of the experiment through the biogenesis pathway. Throughout the pulse-chase assay, three 1 mL aliquots were removed from each culture at each time point. The first aliquot was added to a tube containing trichloroacetic acid , and placed on ice. The second aliquot was added to a tube containing proteinase K , and placed on ice for 20 min to digest Pet exposed on the cell surface. The protease reaction was stopped by the addition of 2 mM phenylmethanesulfonyl fluoride (PMSF) before TCA precipitation. All TCA precipitated samples were washed with acetone, dried, and resuspended in 100 μL of SDS-PAGE loading buffer per 1 OD600 units of cells . Where necessary, a fourth aliquot was treated with 200 μg mL−1 polymyxin B for 10 min on ice prior to proteinase K treatment and TCA precipitation. Ten microliters of the normalized samples were separated by SDS-PAGE on 3–14% gradient gels and subjected to immunoblotting using anti-Pet passenger domain antibodies at a 1:5,000 dilution. These antibodies have been described and validated previously34.The pBAD expression system600 of cultures were normalized (to permit comparison of secreted protein levels) and pelleted before filtering of the supernatant fractions through 0.22 μm pore-size filters. A 10% (w/v) final concentration of TCA was then used to precipitate the secreted proteins as described above. Samples were separated by 10% SDS-PAGE and detected by staining with Coomassie Brilliant Blue R250. Whole cell lysates were separated by SDS-PAGE on 3–14% gradient gels and subjected to immunoblotting using anti-Pet β-barrel domain antibodies at a 1:5,000 dilution as described above. The Pet and EspP β-barrel domains are so similar that these antibodies cross-react with the EspP β-barrel domain. These antibodies have been described and validated previously57.Bacterial cultures harbouring pBADEspP and pBADEspPΔL5 were resuspended in arabinose for 30 min to induce EspP and EspPΔL5 expression. The OD34. Briefly, 40 μg of total membranes were mixed with SDS-PAGE loading buffer and then heated at temperatures between 25 °C and 90 °C. Samples were separated by SDS-PAGE on 3–14% gradient gels and subjected to immunoblotting using anti-Pet β-barrel domain antibodies as described above.After pulse-chase protein expression, cells were harvested and prepared for heat modifiability experiments as described previously34. Briefly, E. coli BL21 (DE3) cells transformed with pETPetΔ1-554 and pETPetΔL5Δ1-554 were harvested  after a 4 h (pETPetΔ1-554) or 18 h (pETPetΔL5Δ1-554) induction with 0.5 mM IPTG. Inclusion bodies were isolated by centrifugation  and solubilized in 8 M urea. PetΔ1-554 and PetΔL5Δ1-554 were then refolded from the urea extracts by rapid 10-fold dilution into tris-buffered saline containing 0.5 % (w/v) lauryldimethylamine-oxide (LDAO) detergent micelles at 35 °C. Affinity purification using Ni-NTA resin was used to separate the refolded passenger and β-barrel domains. Addition of Bio-BeadsTM SM-2 Resin (BioRad) at 0.1 g mL−1 to the unbound flow-through containing the cleaved passenger domain removed the LDAO detergent and residual β-barrel. Subsequent gel filtration chromatography in 50 mM Tris pH 8.0, 150 mM NaCl  resulted in purification of the ~ 51 kDa passenger domain. Bound β-barrel was eluted from the Ni-NTA resin and further purified by gel filtration chromatography in 50 mM tris pH 8.0, 150 mM NaCl, 0.05% LDAO (wt/vol) , which resulted in purification of the folded ~ 30/27 kDa forms.Protein expression and refolding were carried out as previously describedΔ1-554 and PetΔL5Δ1-554 refolding were initiated as described above and were followed by limited treatment with trypsin, which was performed as described previously34. Briefly, aliquots of the reaction mixture were removed at the time points indicated and treated with trypsin (20 μg mL−1) for 20 min on ice before quenching digestion and folding with 2 mM PMSF and SDS loading buffer, respectively. The resulting trypsin fragments were separated by SDS-PAGE and subjected to immunoblotting using anti-passenger and anti-β-barrel domain antibodies as described above.Kinetics of trypsin cleavage profiles during Pet−1 of purified protein were measured at room temperature with a 1 mm cuvette, 1.0 nm bandwidth, 1 s integration time, and 20 nm/min scanning speed. Three scans were averaged, and the spectra were corrected for buffer contribution. For thermal denaturation measurements, the denaturation of β-sheet structure was monitored in the presence of 1% SDS at 218 nm as a function of temperature from 25 °C to 95 °C at a temperature change of 1 °C min–1. Thermal unfolding was irreversible. In both cases, the CD spectra were normalized to the mean residue molar ellipticity.Far-UV CD spectra were collected from 190 to 260 nm using a Chirascan spectrometer. Samples containing 0.1 mg mL−1 of purified protein were collected at room temperature with a 1 cm cuvette, 5 nm slit width, 0.1 s integration time, and an increment of 1.0 nm. Three scans were averaged, and the spectra were subtracted for buffer contribution.A Varian Cary Eclipse fluorimeter was used to collect emission spectra between 300–400 nm upon excitation of tryptophan at 295 nm. Samples containing 0.01 mg mLAll data generated or analysed during this study are included in this published article and its Supplementary Information files. The raw image files of the cropped immunoblots and Coomassie-stained gels displayed in the Figures and Supplementary Figures can be found in Supplementary Fig. Supplementary Information(PDF 4611 kb)Peer Review File(PDF 653 kb)


It is a great challenge to improve the strength of disc superalloys without great loss of plasticity together since the microstructures benefiting the strength always do not avail the plasticity. Interestingly, this study shows that the trade-off relationship between strength and plasticity can be broken through decreasing stacking fault energy (SFE) in newly developed Ni-Co based disc superalloys. Axial tensile tests in the temperature range of 25 to 725 °C were carried out in these alloys with Co content ranging from 5% to 23% (wt.%). It is found that the ultimate tensile strength (UTS) and uniform elongation (UE) are improved synchronously when microtwinning is activated by decreasing the SFE at 650 and 725 °C. In contrast, only UTS is improved when stacking fault (SF) dominates the plastic deformation at 25 and 400 °C. These results may be helpful for designing advanced disc superalloys with relatively excellent strength and plasticity simultaneously. Since the development of aviation industry and power generation needs higher thermal efficiency and lower emission of CO2 and NOx, it is an urgent task to elevate the service temperature and improve the high temperature mechanical properties of the Ni-base superalloys. In the last decades, much attention has been paid to develop advanced superalloys with better strength and plasticity following the strengthening mechanisms such as solution strengthening, work hardening, grain refinement and precipitation strengthening3. These mechanisms are all based on the blocking effects of the defects against dislocation motion, which ranges from zero-dimensional solute atom to three-dimensional precipitated phase4. However, some inevitable shortcomings are also introduced by these strengthening mechanisms, such as element segregation, rapid dislocation recovery and grain boundary oxidization, which will counteract the strengthening effect and diminish the plasticity6. Though increasing γ′ content and decreasing γ′ size can improve the strength in superalloy, the plasticity and hot workability will deteriorate consequently when the γ′ content exceeds 50%7. Considering the limits of these conventional approaches, developing an alternative way to enhance the strength without the loss of ductility is becoming extremely important.Ni based superalloys are broadly used as the hot end components in aircraft engines and land-based gas engines for their optimum matching of mechanical properties, such as excellent high-temperature strength, oxidation resistance, creep resistance, and fatigue properties9. Subsequently, it is confirmed that nanoscale deformation twins (DTs) can also be introduced to strengthen metallic materials either by deformation at extremely high strain rate at low temperature11, or by deformation at moderate level of strain with low-SFE12 when dislocation activities are effectively suppressed. Though the severely deformed metals and alloys can yield a great promotion in strength, they will inevitably result in an obvious drop in ductility, exhibiting a typical “banana-shaped” strength-ductility trade-off relation due to the limited capacity of strain hardening of the nanograins embedded with nanotwin bundles15. Better strength-ductility synergy can be obtained by applying annealing treatment on the severely deformed materials, in order to introduce some larger recrystallized grains that producing extra ductility with sacrificing some strength16.A novel strengthening mechanism has been discovered in the ultrafine-grained Cu that nanoscale growth twins can induce remarkable strengthening and toughening effects by acting both as dislocation blockers and dislocation slip planes19. With the decrease of SFE, wavy-slip will be gradually replaced by planar-slip, because a large separation between partial dislocations induced by lowering SFE inhibits their recombination and extension on the cross-slip plane, causing dislocations to organize themselves into planar arrays or planar slip bands21. Apart from dislocation substructures, the twinning tendency can also be promoted obviously due to the reduced critical stress of deformation twinning with decreasing SFE25. Under tensile deformation, wavy slip and dislocation cell are prevailing in pure Cu, while planar slip and deformation twinning will be activated in the alloyed Cu with lower SFE19. It has been demonstrated that this kind of DTs induced by quasi-static deformation cannot only improves the strength, but also enhances the plasticity, creating a unique effect of synchronous improvement of strength and plasticity in Cu-Al alloys26. The underlying mechanisms responsible for the SISP are well analyzed on the basis of the intrinsic features of DT and its critical role in the plastic deformation26. The improved strength is attributed to that DTs can act as obstacles for dislocation motion and help to hinder the dislocation recovery by separation them into smaller regions. The improved plasticity is ascribed to that the deformation twinning can improve deformation homogeneity due to the enhanced strain-hardening ability, and the twin boundaries can serve as the dislocation slip planes to accommodate plastic strain. Up to now, the existence of SISP has been confirmed in some other alloy systems such as Cu-Zn18, stainless steels27, which shares the commonality that adding an alloying element to decrease SFE, driving the deformation twinning to dominate plastic deformation.The role of SFE in the deformation behaviors of face-centered cubic (fcc) metals has been extensively studied in Cu and Cu alloys with wide variation of SFE by altering Al or Zn content30. The prominent differences between MT and DT lie in the twin thickness and twinning mechanisms, the MT has smaller thickness with only 4–50 atom layer, and more complicated twinning mechanism that involving atom diffusion and reordering in a thermally-activated process. However, the influence of MTs on the mechanical properties of crept alloys are scarcely mentioned, because only small content of MTs can be produced at the high-SFE materials. Enhancing the twinnability of superalloys via decreasing SFE has been proved to be practicable, substantial deformation MTs can be introduced during deformation at intermediate temperatures (650–800 °C)34. It is suggested that these MTs could largely improve the tensile strength and creep life of the tested alloys. Actually, it is hard to give an explicit evaluation on the contribution of MTs to mechanical properties, because the change of SFE is always accompanied with the variation of solution strengthening elements, γ′ forming elements and even the grain size. These factors inevitably affect the deformation mechanism and mechanical property, so the improvement in mechanical property cannot be only attributed to MTs without considering the disturbance of other factors.However, it is worth noting that the SISP introduced by deformation twinning is only discovered in the binary alloys or single-phase alloys subjected to deformation at ambient temperature. While for the multiphase-structured superalloy containing complicated chemical compositions, DTs can hardly form during plastic deformation at ambient temperature according to the published results. Instead, a particular mechanism named as microtwinning has been reported frequently that always operates during the deformation at high temperature and low strain rate such as creep, leading to the formation of microtwins (MTs) in superalloys3-typed γ′ phase. Adding the Co content can effectively increase the content of electron holes, results in the decrease of SFE in Ni-Co based disc superalloys35. The SFE values of 5Co, 15Co and 23Co alloys at room temperature were calculated to be 40.1, 33.3 and 24.9 mJ/m2, respectively, via measuring the separation width of two partials of the stacking faults in γ matrix36. So, the calculated SFE value actually belongs to the γ matrix of tested superalloys, representing their twinnability during deformation. According to the previous result37, the value of SFE is temperature-dependent, increasing temperature can lead to the nonmonotonic change of SFE in superalloys. In spite of this, it is reasonable to deduce that the decrease of SFE by adding Co element may not be affected by temperature on account of the strong effect of Co element on SFE. Besides the composition design, these alloys were then treated in the same hot working and mechanical processing to make sure that they have nearly the same phase constitution, similar grain size, and negligible discrepancy of γ′ volume fraction and size.Consequently, it is necessary to eliminate these factors in order to get better understanding on the imposed influence of SFE both on the microscopic mechanisms and macroscopic properties of superalloys. Considering that, three newly developed Ni-Co based disc superalloys were chosen as experimental alloys, with Co content increased from 5 wt.% to 23 wt.% by substituting Ni without the change of other alloy elements, for the sake of ensuring the SFE being merely related to Co element. The added Co element in Ni-based superalloys will preferentially partial to the γ matrix, others enter into the γ′ phase by substituting Ni atom, forming 38. The nearly unchanged volume fraction of γ′ precipitates with increasing Co content can be rationalized that the three superalloys contain the same γ′-forming elements, i.e. Al and Ti. Generally, increasing Co content can enhance the stability of precipitates and lower the γ′ solvus temperature, which widens the processing window and reduces the thermal stresses induced by controlled cooling and quenching in superalloys31. It has been reported that high Co addition up to 30% made no difference to the phase constituent, but the size of secondary γ′ will firstly decrease with increasing Co content, and then remain unchanged with further addition of Co above 25%39. Since higher Co content will result in lower γ′ solvus temperature in alloys, in this case secondary γ′ will form at lower cooling temperature and have less time to grow up. But in our study, it can be found that Co content has unconspicuous effect on the size of γ′ precipitates due to the same content of Al and Ti elements. Based on the above results, it can be inferred that the three alloys have comparable grain size, strengthening phase constitution and distribution, the discrepancy induced by Co variation can be roughly neglected, which gives us a chance to separately investigate the influence of SFE on mechanical behaviors without the other disturbances.The original microstructures of the three alloys before tensile deformation are depicted in Fig. The tensile stress-strain curves of three alloys during plastic deformation at temperatures ranging from 25 °C to 725 °C are depicted in Fig. When deforming at higher temperature, the three alloys exhibit distinct mechanical behaviors compared with that at 25 and 400 °C. Firstly, the influence of Co content on UTS becomes much more evident, reflecting in larger increasing magnitude of UTS with increasing Co. For instance, the UTS can be increased by 87 MPa and 153 MPa at 650 °C, while it can be increased by 138 MPa and 89 MPa at 725 °C in response to the addition of Co content from 5% to 15% and from 15% to 23%, respectively Fig. . Secondl26. It can be deduced that SISP happened in these Ni-Co based alloys is also resulted from the modified microscopic deformation mechanism that being benefit for work hardening. While, unlike the effect of Co content on the tensile properties, the increase of deformation temperature will reduce the strength and elongation of tested superalloys simultaneously, which is different from the tradition FCC metals that the tensile strength will decrease and the elongation will increase with the increase of temperature. This can be attributed to the activation and acceleration of oxidation introduced by increasing the deformation temperature, which will result in the local cracking of grain boundaries and the deterioration of ductility.More intuitive dependence of UTS on UE under different Co content and temperatures are plotted in Fig. 2  deformed at temperature up to 725 °C, nevertheless dislocation pairs, SFs and MTs will be triggered respectively in the temperature range of 25–650 °C, 650–700 °C, and 700–725 °C for the TMW-4M3 alloy with SFE of 19.9 mJ/m2 31. The prominent discrepancies in the influence of SFE on the deformation mechanism between the present three alloys and other disc superalloys may derive from the different alloying systems.In order to figure out the underlying mechanisms that causing SISP, microstructural observation was carried out to detect the deformation behaviors of the tested alloys under different conditions. It is clearly revealed that there are two distinct mechanisms controlling the deformation at low temperatures and high temperatures respectively, as depicted in Fig. 40. The transformation of deformation micromechanisms is not only temperature-dependent, but also related to the relatively low SFE of the three superalloys, which can be rationalized by the corresponding formation mechanisms of the SF and MT. According to the previous results, relatively low SFE is the crucial prerequisite for MT formation in superaloys, regardless of whether they were prepared by casting or forging, or strengthened by solution atoms or γ′ precipitates when deformed at intermediate temperatures41. It is evident that two distinct deformation mechanisms are in good agreement the two different mechanical behaviors of three alloys, indicating the different role that played by SF and MT in the strength and plasticity of superalloys. The contribution to mechanical properties made by SF and MT will be discussed in the following section on the basis of their characteristics variation with SFE.It is shown that the transformation of deformation micromechanisms from SF shearing to deformation microtwinning will occur in the three superalloys in response to the increase of deformation temperature from 400 °C to 650 °C. The improvement in twinning ability with increasing temperature is a unique characteristic in these superalloys, since twins are more prone to form at low temperature or high strain rate in many other metals2 to 24.9 mJ/m2 at 725 °C. However, the SF spacing just decreases from 12.9 nm to 6.6 nm, and the SF length only increases from 80.4 nm to 111.7 nm under the same SFE range at 400 °C. Besides, the dependence of MT spacing and length on temperature is much more apparent for the three alloys: higher temperatures lead to larger MT spacing and MT length.It is widely accepted that SFE plays a significantly role in the nucleation and growth of SFs and MTs. Considering that the thickness of the MT in this study is usually less than 50 atom layers, the change in the thickness with SFE will be neglected, and we only emphasize on the changes in the spacing and length of the SFs and MTs. Statistical calculation was carried out on the variation of SF/MT length, spacing and areal density with SFE in the three alloys, as exhibited in Fig. The areal density of SF/MT, defined as the total length of SF/MT in a unit area, is carefully measured under different temperatures. Figure 42, prismatic glide mechanism43, and faulted dipole mechanism44 according to the characteristics of the dissociation process of the 1/2 <110> dislocation. In the γ′ strengthened Ni-base superalloy, the formation of DTs is always associated with the loading at high temperature and low strain rate45. The major differences of twinning procedure between common fcc alloys  and superalloys are that the former is single-phase solid solution, easier to be deformed uniformly, while the latter contains two phases of γ/γ′, deformation always starts firstly in the γ corridors, and is trapped at the γ/γ′ interface. Apart from the externally loading conditions, the size, volume fraction and chemical compositions of the γ′ precipitates can strongly affect the activity of trapped dislocations at the interface, which decide the twinning mechanism of superalloys.Deformation twinning always prevails in the fcc metals and alloys that deformed at the low temperature and high strain rate while dislocation activities are greatly suppressed. For the coarse-grained fcc metals, several well-known twinning mechanisms have been developed to explain the initiation and propagation of the DTs, such as the pole mechanism46. The second is SF overlapping introduced twinning, the 1/3 < 1 1 2 > superpartial dislocations directly penetrate into γ′ precipitates, leaving superlattice intrinsic or extrinsic SFs on the parallel and neighboring/non-neighboring {1 1 1} planes. For instance, high-temperature deformation (compression and creep) in some γ/γ′ structured Co-based alloys will result in the dissociation of perfect dislocations into partial dislocations of 1/3 <1 1 2> (leading) and 1/6 <1 1 2> (trailing). Due to the high SFE of L12-Co3 (~91 mJ/m2) as calculated by first-principles47, SFs are more frequently produced by the consecutively passage of the leading partials within γ′ phase50. With lowering the SFE, deformation MTs may also be introduced in the Co-based superalloys when the overlapping tendency of SF is increased. The third is thermally-activated microtwinning proposed by Kolbe51, twin formation takes place by a diffusion-mediated reordering within γ′ precipitates after the pairwise passage of identical a/6 <112> Shockley partial dislocation shearing across both γ and γ′ on adjacent {1 1 1} planes.Generally, three twinning mechanisms are well-documented for superalloys. The first is zero-strained twinning, it was discovered in a coarse-grained disc superalloys at tensile strain of 3%, this twinning occurs through the cooperative dissociation of three full dislocations or by the overlapping of paired parallel and non-neighboring SFs52. It is shown that reordering as a rate-limiting process requires very simple, vacancy-mediated exchange between Al and Ni atoms, and could be generalized to the formation of superlattice intrinsic and superlattice extrinsic SFs45 in superalloys subjected to the similar deformation condition. Based on the above analysis, the reason for the absence of MTs at RT and 400 °C can be rationalized as the high-energy barrier for the atom reordering in the overlapped CSFs, therefore SFs are prevalent in the low temperature range. According to the third theory, if the SFs in γ′ precipitates are formed through the shearing of Shockley partials, they actually are the unstable CSFs. However, it is also possible that SFs may be ascribed to the running through both of γ and γ′ by the 1/3 <1 1 2> superlattice partial dislocations for the high flow stress at low temperatures53.In this study, deformation at higher temperatures will promote the MT formation, deformation at lower temperatures only produces SFs, this strong temperature-dependent formation of MTs indicates that twinning is likely a thermally-activated process. As is suggested by the third twinning mechanism, when L12-structured γ′ precipitate is sheared by one Shockley partial dislocation, a high-energy complex stacking fault (CSF) will be introduced. The overlapping of these CSFs is probable when a single γ′ precipitate is sheared by one set of Shockley partial dislocations with the same Burgers vector, then pseudo twin will form by the overlapping of at least four neighboring CSFs. These pseudo twins all have an orthorhombic structure with numbers of unstable, high-energy Al-Al nearest neighbor bonds. Then diffusion-mediated atomic reordering need to be provoked between two adjacent CSFs, in order to eliminate the Al-Al bonds, restoring again the initial L12 structure and transforming the pseudo twin to a true MT. This mechanism has been demonstrated by direct TEM observation, theoretical modeling and computer simulation during creep deformation at intermediate temperature25. With the decrease of SFE, room-temperature SISP can be produced in the Cu-Zn18 alloys, Cu-Al alloys26 and stainless steels27. The deformation mechanisms in superalloys may be greatly affected by the intrinsic factors  and extrinsic factors . In superalloys, the deformation micromechanisms are also controlled by its SFE, which may disperse widely on the alloying system and deformation temperature. Generally, the deformation mechanisms of superalloys with medium to high SFE are mainly participated by the dislocation activities. For instance, in the tension deformed René 80 superalloy, γ′ shearing occurs by the slip of APB-coupled 1/2 < 1 1 0 > dislocation pairs on {1 1 1} planes and then cross-slip to {100} planes to act as obstacles to moving dislocations at temperatures below the peak temperature, and then by the viscous slip of two superlattice intrinsic SFs (SISFs)-separated 1/3 < 1 1 2 > superpartials whose cores have split on {1 1 1} planes at temperatures above the peak temperature54. Other researchers found that γ′ shearing by dislocation pairs or slip bands dominated the tensile deformation process of traditional polycrystalline superalloys at 600 °C, nevertheless γ′ by-pass was activated and turned to be the main deformation mechanism when the temperature was increased from intermediate (600–850 °C) to high temperatures (beyond 850 °C) regime55.In some fcc binary or single-phase alloys, such as Cu-Al and TWIP steels, the deformation mechanisms are proved to be controllable. Through decreasing the SFE of alloys, the planarity of dislocation slipping is enhanced greatly, the deformation mode will be changed from cross slip to planar slip, and even to deformation twinningIn this study, the changes of temperature and SFE have prominent effects on the deformation behaviors and mechanical properties of three alloys. Firstly, temperature promotes the transformation of deformation mechanisms from SF shearing to deformation twinning. The introduction of SF and MT in the three alloys is also related to the relatively low SFE. Secondary, though the deformation mode of three alloys has no obvious difference at the same temperature, the spacing and length of SF/MT are greatly affected with decreasing SFE. This can be understood in view of the procedure of SF/MT nucleation and propagation. The spacing of the two planar defects depends on the nucleation capability: when the nucleation capability is enhanced, the spacing will be decreased. It has been clarified that the nucleation tendency of SF is mainly decided by the material characteristics and deformation conditions, such as SFE, temperature and strain rate, etc. In this study, under the same deformation conditions, the difference in SF spacing between three alloys could be ascribed to their distinct SFE. As the SFE decreases, two consequences will promote the nucleation of SF: 1) more perfect dislocations will be disassociated following the response to the external force driving; 2) dislocation recovery will be reduced greatly that results in a higher density of Shockley partials in the deformed microstructure. At the MT dominant temperature, as the SFE decreases, more pseudo twin will be generated and the transformation of pseudo twin to real MT will be facilitated. So, the SF/MT spacing will decrease with decreasing the SFE.56. Under a shear stress, the decomposition and slipping of dislocations exhibited distinct features from the stress-free condition. Firstly, stress can induce more perfect dislocations to resolve by providing necessary energy for dislocation reaction. Secondly, stress will drive the leading dislocation slipping further since the slip of leading dislocation can assume large portion of strain. In the present study, stress and SFE play a great role in the formation of SF/MT. Alloy with relatively low SFE has lower interfacial energy that may lead to the leading partial slipping further, so longer SF/MT will be generated.Under a stress-free condition, a perfect dislocation in fcc crystalline structure will resolve and form an extended dislocation in order to reduce the energy, which consists of two partials that embedded with an SF ribbon. The spacing of two partials is a balancing of attractive force and expulsive force. The length of extended dislocation is decided by the slipping distance of the leading partial: farther distance results in larger length. It has been demonstrated that the effective force, Orowan stress and SFE have great effects on the slipping of the leading Shockley partial dislocation57. However, the UE changes little with the variation of SFE, especially at 25 °C, showing that SFE has weak influence on the UE. This should be ascribed to the limited capacity of storing dislocations in the SF structure with only two or three layers of atoms. In contrast, at the MT operating temperatures, decreasing SFE can increase the density of MTs at a higher amplitude of variation. Previous results based on molecular dynamics simulations showed that a screw lattice dislocation may either propagate into the adjacent twin grain by cutting through the boundary, or dissociate within the boundary plane when confronted with the coherent twin boundary forced by the external stress58. While a non-screw dislocation will dissociate into different partial dislocations penetrating into the twin and/or gliding along the twin boundary, producing a sessile dislocation lock at the twin boundary as well59. Accordingly, the increased density of MTs will produce more obstacles for dislocation motion, and supply more glide planes to accommodate dislocation motion, resulting in a synchronously improvement of strength and plasticity (SISP) in the Ni-Co based superalloys.The above results clearly demonstrate that introducing planar defect of MTs by decreasing SFE can produce SISP for disc superalloys. As summarized in Fig. Decreasing SFE is a crucial prerequisite for promoting SISP in disc superalloys, it is successfully achieved by altering Co content in the Ni-Co based disc superalloys. It may also be obtained by adding other single or multiple alloying elements, including W, Mo for solution strengthening, and Al, Ti for precipitation strengthening, and C, B interstitial atoms for GB strengthening, and Cr for improving oxidation resistance, which need further studies in future. According to the author’s knowledge, Co is an alloying element that significantly affects the SFE and nearly does nothing to harm the mechanical properties of tested alloys when its content is below certain value. In addition, since the MTs are generated at the intermediate temperatures, this strengthening mechanism also takes place in the same temperature range, which is just the common service temperatures of the disc superalloys. This finding gives a powerful illustration and attractive prospect of strengthening and toughening nickel-base disc superalloys by mediating SFE.2). The specimens for SEM were electronically etched in a solution of 80 ml H2O +5 ml glacial acetic acid +15 ml nitric acid solution at 1.5 V, for 30 s.The tested alloys were casted as 20-kg ingots by vacuum induction melting following the nominal composition of 5/15/23Co-14Cr-2.8Mo-1.2W-5.6Ti-2.3Al-0.02B-0.02C-0.03Zr-Ni balance (Wt.%). For convenience, the alloys containing 5%, 15% and 23% Co were labelled as 5Co, 15Co and 23Co, respectively. After homogenizing treatment to alleviate composition segregation, these ingots were hot extruded into ϕ35 mm billets with extrusion ratio of 6 at about 1140 °C. The extruded billets were subsequently solution treated at 1100 °C for 4 hours followed by air cooling (A.C), then aged at 650 °C for 24 hours followed by A.C, and finally aged at 760 °C for 16 hours followed by A.C. Dog-bone samples for tensile test were machined into the size of M6 mm × 45 mm with gauge section of M3 mm × 15 mm along longitudinal direction of alloy bars. Before tensile test, all the specimens were electropolished to eliminate the scratches in an electrolyte of 10 pct. perchloric acid and 90 pct. ethanol under 11 V for 1 min at room temperature. The specimens for optical microscopy (OM) observation were etched in a solution of modified Kalling reagent (100 ml HCl, 100 ml methanol and 50 g CuCl−4 s−1. At least two specimens were tested under each condition to ensure the data reliability. Specimens were kept in electric furnace for 15 min under each setting temperature before test to eliminate temperature fluctuation on the gauge part. Tensile strain was monitored by an extensometer with full range of ±20%. The microscopic mechanisms for tested specimens were investigated using a FEI Tecnai F20 TEM operated at 200 kV. Disks with a thickness of ~500 μm for TEM were cut using electrical discharge machining from the samples perpendicular to the stress axis. The disks were manually ground down to 50 μm and then perforated by a twin-jet electro-polisher in a solution of 10% perchloric acid and 90% ethanol at about 31 V and −22 °C.Tensile tests were performed on an Instron 8862 machine at temperatures ranging from ambient temperature to 725 °C with a constant strain rate of 3 × 10


Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the T. pallidum peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in T. pallidum we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside Treponema. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the T. pallidum cell envelope. Treponema pallidum subspecies pallidum  is a medically important spirochete bacterium and the causative agent of syphilis, a chronic, multistage systemic disease transmitted sexually, through direct contact with an infectious lesion, or in utero. It is estimated that the current global burden is 36 million cases with 11 million new infections each year ) and GC GGC CGC ATC GCG CAA GAT GGT GAT CTC C5´-CTG TCT G  (antisense primer with NotI restriction site [underlined]). The PCR amplicon was then cloned into a modified pET28a  vector with a TEV protease cleavable N-terminal hexa-histidine tag. The construct was confirmed by DNA sequencing. The construct was transformed into the E. coli expression strain BL21 Star (DE3) (Invitrogen). Transformants were cultured at 37°C in LB broth containing 100 μg/mL ampicillin until the A600 reached 0.4. Cultures were induced with 0.4 mM IPTG for 16 h at 25°C and harvested by centrifugation at 4°C. Cell pellets were resuspended in buffer  and flash frozen in liquid nitrogen and stored at -80°C.Eight signal sequence prediction servers were used to predict the presence and position of a potential cleavable signal peptidase type I (SPI) signal sequence within the N-terminus of Tp0624: PrediSi  pre-equilibrated in HEPES-buffered saline . Notably, Tp0624 eluted from the SEC as a monomer (Purification of recombinant protein was performed using immobilized nickel affinity and size exclusion chromatography (SEC). Briefly, recombinant His-tagged protein was loaded onto 1 ml HisTrap FF affinity columns  pre-packed with pre-charged Nickel Sepharose 6 Fast Flow resin. Columns were then washed with buffer  and bound recombinant protein was eluted in a buffer consisting of 20 mM HEPES pH 7.5, 500 mM NaCl, and 500 mM imidazole. Tp0624 was then further purified by SEC on a HiLoad monomer . The N-t2, 14% PEG3350) and were equilibrated against 300 μL of reservoir solution. Crystals appeared within 24 h and grew to a final size within 3 days. Crystals were cryoprotected in reservoir solution supplemented with 12.5% glycerol and flash cooled in liquid nitrogen. Diffraction data were collected on beamline 08ID-1 at the Canadian Light Source (CLS). For phase determination, Tp0624 was crystallized using the same technique as for the native crystals but the reservoir solution consisted of 0.1 M HEPES pH 6.8, 0.45 M MgBr2, 14% PEG3350. Diffraction data were collected on beamline 08ID-1 at CLS using an optimized wavelength of 0.9201 Å for the f” bromine edge.Crystals of Tp0624 were originally identified in the MCSG-1 screen (Microlytic) using sitting drops at 18°C. The final, refined drops were set in hanging drops and consisted of 3.6 μL Tp0624 at 10 mg/mL in HBS with 1.8 μL of reservoir solution  or 1.92 Å (Tp0624-Br) resolution using Imosflm  and AimlT. pallidum query sequence  and the NCBI non-redundant database. Tp0624 residues A64-F177, Y198-C317, and D332–D476 were used for the identification of orthologous proteins containing domains with significant homology to Tp0624 domains 1, 2, and 3, respectively. All identified orthologs had an e-value cutoff of 0.00026.Orthologs of Tp0624 were identified using BLASTp , the T. pallidum isolated from an infected rabbit. Somewhat unexpectedly, we identified a peptide that mapped to the predicted signal sequence region of Tp0624 . Th. Th2 at  with D2 . While LThe C-terminal domain, D3, which is classified based on sequence to be part of the OmpA-OmpF porin (OOP) family , is the E. coli Outer Membrane Protein A (OmpA), which contributes to cell envelope stability by providing a structural link between the peptidoglycan layer and outer membrane [Vibrio PomB, an inner membrane-anchored MotB ortholog and component of the flagellar motor that anchors the stator to the peptidoglycan layer . The function of all OmpA-like domains that have been characterized to date is centered on their coordination of peptidoglycan via a pocket at the base of the domain [Acinetobacter baumannii OmpA-like domain, PDB ID 3TD5) reveals key differences between Tp0624 D3 and canonical OmpA-like domains. For example, a loop that forms the outer border of the peptidoglycan binding site in other OmpA-like domains is displaced by nearly 15 Å in Tp0624 (D(x)7NxxLSxxRA, with Asp2, Asn10, and Arg17 being the most critical and highly conserved binding residues [AD(x)6E/QxxLSxxRA motif ; despite a sequence identity of only 4%, these domains overlay with an rmsd of 3.3 Å over 79 positions, corresponding to a Z-score of 5.4. While the central four-stranded beta-sheet and primary helix are well conserved, both ends are structurally divergent with apparent functional consequences. Specifically, the presence of helices at the base of Tp0624 D1 uniquely enable the domain to pack against D2 and D3, while the opposite end of D1 is truncated compared to the trigger factor where it is used to engage the ribosome [In contrast to D3, D1 and D2 do not show significant sequence identity to any proteins of known structure or function. Structural analysis of D1  using thribosome . These dribosome , to a plribosome .Plasmodium 6-Cys and Toxoplasma SRS domains [In striking contrast to the more than 1200 structural similarity hits for Tp0624 D1, no structural analogues of D2 were identified. Thus, the Tp0624 D2 fold can be described as a previously uncharacterized domain. The defining feature of this domain is its orthogonally packed four-stranded beta-sheets flanked on three sides by four alpha-helices . The ort domains –49. ThisThe intriguing modularity and domain architecture of Tp0624 indicate the possibility for functional interdependence, which led us to ask whether all three domains are reliant on each other for function or if specific domains are likely to act independently. To address these questions, we analyzed bacterial sequence databases using the domain boundaries established from the high-resolution structure . We founT. primitia, T. azotonutricium, and T. caldaria), suggesting that D3 can augment a treponeme-specific function of the D1-D2 pair. Next, we assessed the evolutionary relationship between the Tp0624 treponemal orthologs using full-length sequences or the three individual domains  [T. pallidum possesses a MotB homolog (Tp0724) within a flagellar operon [Based on the data reported here, we propose two models describing potential subcellular locations and corresponding functional peptidoglycan binding roles for Tp0624 . In the otein M) , 9 to prr operon  it is per operon . Given tr operon ; Tp0624 T. pallidum has a strategically minimalized complement of proteins, yet is one of the most invasive pathogens. Key virulence traits of T. pallidum include its unusual peptidoglycan structure, fragile cell envelope and internal endoflagella, yet the molecular strategies to functionalize and stabilize these ultrastructural features are largely unknown. In this study, we determined the high-resolution structure of Tp0624, and showed it to be comprised of a modular, three-domain architecture incorporating an OmpA-like domain and a tandem pair of previously uncharacterized domains. Intriguingly, the OmpA-like domain (D3) is sufficiently divergent that it is unable to bind the conventional DAP peptidoglycan component and may be uniquely adapted to coordinate the novel peptidoglycan structures of T. pallidum and potentially rigidify and stabilize the cell envelope. While we have not been able to establish a function for the previously uncharacterized domain pair (D1-D2), its association with D3 appears to correlate positively with treponeme pathogenicity and thus may play an important role in supporting the function of D3. Overall, our data suggests that Tp0624 may coordinate peptidoglycan through an unconventional mechanism to stabilize the inner- or outer-membranes of the highly divergent human pathogen, T. pallidum.S1 FigSuperdex 75 HiLoad size exclusion chromatogram showing that Tp0624 (46 kDa) elutes as a monomer (elution peak of 44 kDa globular standard denoted by hash mark).(PDF)Click here for additional data file.S2 FigOrthologs of Tp0624 were identified using BLASTP  and aligned in Clustal Omega and BioEdit sequence alignment editor. The predicted Tp0624 signal peptide cleavage site at T61-Q62 (SP), domains 1 (D1), 2 (D2), and 3 (D3), known OmpA domain peptidoglycan binding residues (TD(x)7NxxLSxxRA) and corresponding treponemal residues (red rectangles) are shown. Identical residues (black background), similar residues (gray background), and dissimilar residues (white background) are highlighted. At each position of the consensus sequence, an asterisk indicates full conservation among the 18 treponemes, a colon indicates strong conservation, and a dot indicates lower conservation.(PDF)Click here for additional data file.S3 FigSpirochaeta thermophila (Accession number: WP_013314620). The dashed line indicates the phylogenetic divide between pathogens (top) and non-pathogens (bottom).A phylogenetic tree of 18 full-length Tp0624 ortholog sequences from treponemes was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was calculated. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method and were in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted using MEGA  6 software. The tree was rooted with the Tp0624 ortholog from (PDF)Click here for additional data file.S4 FigSpirochaeta thermophila (Accession number: WP_013314620). Pathogens (P) and non-pathogens (NP) are indicated.A phylogenetic tree of Tp0624 ortholog sequences corresponding to domain 1 from 18 treponemes was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was calculated. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method and were in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted using MEGA  6 software. The tree was rooted with the Tp0624 ortholog from (PDF)Click here for additional data file.S5 FigSpirochaeta thermophila (Accession number: WP_013314620). Pathogens (P) and non-pathogens (NP) are indicated.A phylogenetic tree of Tp0624 ortholog sequences corresponding to domain 2 from 18 treponemes was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was calculated. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method and were in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted using MEGA  6 software. The tree was rooted with the Tp0624 ortholog from (PDF)Click here for additional data file.S6 FigSpirochaeta alkalica (Accession number: WP_018525408). Pathogens (P) and non-pathogens (NP) are indicated.A phylogenetic tree of Tp0624 ortholog sequences corresponding to domain 3 from 15 treponemes was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was calculated. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method and were in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted using MEGA  6 software. The tree was rooted with the Tp0624 ortholog from (PDF)Click here for additional data file.


Helicobacter pyloriββαβββMicrotubule Affinity-Regulating Kinase 2 (MARK2) protein has a substantial role in regulation of vital cellular processes like induction of polarity, regulation of cell junctions, cytoskeleton structure and cell differentiation. The abnormal function of this protein has been associated with a number of pathological conditions like Alzheimer disease, autism, several carcinomas and development of virulent effects of  In the majority of AMPK subfamily members, kinase core, UBA and KA1 domains are conserved ones . ActivatUBA domain is a globular domain of 40 residues arranged mainly in three α-helices. It is known as the UBA domain due to its sequence homology to the class of ubiquitin-associated proteins . HoweverHere, we modeled the inactive core structures of native and T208E mutated forms of human MARK2 protein, covering residues 49-363. It has been reported that this mutation increases the kinase activity by four fold . Both moModeling: In order to construct the wild type MARK2 and mutant MARK2T208E structures, MODELLER software version 9.9 [Rattusnorvegicus, but have exactly the same sequence as that of Homo sapiens in our target area of study  with τT = 0.1 ps [P = 0.2 ps during which density of the system was stabilized at around 1000 kg/m3 [on 4.5.5 , with Amon 4.5.5 . The sta= 0.1 ps . After a00 kg/m3 . Finally00 kg/m3 . Lennard00 kg/m3 . The neiFree energy calculations: Free energy (∆G) of interaction between UBA domain and protein N-lobe was calculated using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. 400 frames extracted from the last 8000 ps of each trajectory corresponding to wild and mutant structures were used for analysis. Calculations were performed with the scripts kindly provided by Dimitrios Spiliotopoulos [topoulos .Quality of models: Among the 1000 models generated by MODELLER for MARK2 structure, the one corresponding to the lowest value of the energy and Dope score was selected and quality of the model was evaluated by ERRAT [by ERRAT  and WHATby ERRAT  softwareby ERRAT . We alsoMD simulation: Both MARK2 and MARK2T208E models were subjected to 20 ns of MD simulations. The backbone root mean-square deviation (RMSD) of MARK2 and MARK2T208E structures relative to their own starting structures were 1.75 and 2.39 Å, respectively. As seen, average RMSD of MARK2T208E is higher than that of the native one, indicates significant conformational rearrangement in MARK2T208E caused by the activating mutation. For both models, the backbone RMSD as a function of time reaches a relative plateau after about 12 nanoseconds (ns) of simulation (data not shown) and from this time point on, motions have been studied (data not shown). Comparison of RMSD values in the two lobes of the protein implies that gross deviations chiefly happen in the N-lobe of protein and activation segment  that is proved to have a key role in regulation of numerous kinase proteins activity [T208E structure compared to that of MARK2. This is also the case for αC-helix (residues: 92-105) (T208E shows that the N-lobe of the protein has rotated by about 10 degrees toward the C-lobe. This rotation is accompanied by a decrease in the average distance between β1 and β6 sheets (from 17.33Å to 12.51Å) which causes further tightening of the ATP entrance site. As is expected due to T208E mutation, beta sheets of the N-lobe are dragged toward the kinase active site, but nevertheless RMSD measurements indicate that movements of β1-β4 sheets toward the active site are not uniform and UBA-neighboring parts have been less deviated from the starting structure in comparison with those of having no direct interaction with this domain. In the case of wild type (MARK2) structure, RMSD pattern does not show an ordered trend like that of MARKT208E structure (activity . Throughactivity . DSSP an 92-105) . Comparitructure .T208E average structures revealed that although this motion of αC-helix is set out in MARK2T208E structure, but again like that of beta sheets, this expected motion toward the protein active site is not uniform in all through the αC-helix structure , αF-helix (residues: 230-240) and α1-helix of UBA domain (residues: 329-335) is more intense within the mutant protein compared to wild structure . Detailed analyses imply that dragging movements of UBA and N-lobe domains toward the protein mass center in MARK2T208E structure have been compensated by simultaneous expansion of UBA domain, so that the total protein volume remains almost constant in mutant structure.tructure . This catructure . FurtherModulation of enzymatic activity through an extension situated to the C-terminal of catalytic core which wraps around the core domain of enzyme is a common regulatory mechanism in many protein kinases . In MARKInduction of T208E mutation and studying the protein structure with small-angle X-ray scattering (SAXS) analysis by Marx and colleagues showed that the UBA domain is attached to the N-lobe during the study and no significance difference in protein compactness was observed through the analysis . RegardiIn a separate study, Jaleel and colleagues came to different results by showing that the enzymatic activity of MARK2 was strikingly reduced after omission of UBA domain . Their rThere are also reports indicating that the activity of MARK2 phosphorylated by LKB1 is about 20 times higher than that phosphorylated by MARKK , 11. On ), but the UBA domain applies a mild break to this trend and makes the MARK2 activation dependent on phosphorylation of the conserved threonine (Thr208) of the activation loop. The extent of rigidity within the UBA or UBA-like domains may also contribute to the level of auto-inhibition. The sequence of this domain is not highly conserved among all members, but all have a similar 3D structure consisting of three alpha helices . There is also a conserved glycine residue within the connecting loop of α1 and α2 helices (Gly336 in MARK2) [There is also another reason to attribute a mild auto-inhibitory function to the UBA domain; Those RD protein kinases that need phosphorylation on activation segment to fulfill their activity, have a basic residue like arginine or lysine on β9 strand which contributes to the formation of RD pocket. Substitution of these residues with neutral or acidic ones, emancipate the kinase from phosphorylation dependency for activation , 30. In n MARK2) . In MARKn MARK2) . Confirm