However, relatively few studies have extensively examined the roles of individual residues and domains of globin-coupled diguanylate cyclases, which can shed light on their signaling mechanisms and provide drug targets. Here, we report the critical residues of two globin-coupled diguanylate cyclases, EcGReg from Escherichia coli and BpeGReg from Bordetella pertussis, and show that their diguanylate cyclase activity requires an intact globin domain. In the distal heme pocket of the globin domain, residues Phe42, Tyr43, Ala68 (EcGReg)/Ser68 (BpeGReg), and Met69 are required to maintain full diguanylate cyclase activity. The highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg/BpeGReg are essential to diguanylate cyclase activity. We also identified sixteen important residues  in the active site and inhibitory site of the diguanylate cyclase domain of EcGReg. Moreover, BpeGReg266 (residues 1–266) and BpeGReg296 (residues 1–296), which only contain the globin and middle domains, can inhibit bacterial motility. Our findings suggest that the distal residues of the globin domain affect diguanylate cyclase activity and that BpeGReg may interact with other c-di-GMP-metabolizing proteins to form mixed signaling teams.Globin-coupled diguanylate cyclases contain globin, middle, and diguanylate cyclase domains that sense O The Ne pocket , 8–10. Ie pocket , 8.C-di-GMP-dependent signaling pathways regulate diverse cellular functions including motility, biofilm formation, virulence, differentiation, and the cell cycle . VariousEscherichia coli, Bordetella pertussis, Azotobacter vinelandii, Desulfotalea psychrophila, Shewanella putrefaciens, and Pectobacterium carotovorum [EcGReg  from E. coli and BpeGReg from the whooping cough pathogen B. pertussis, can sense O2 to regulate c-di-GMP synthesis [2 affinity and cooperative PDEs [EcGReg and Ec DosP (EAL type) couple to control c-di-GMP homeostasis, whereas BpeGReg may cooperate with Bpe RpfG (HD-GYP type) [EcGReg)/Ser68 (BpeGReg), and Met69, which are in the distal heme pocket of the globin domain. We also tested the highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg and BpeGReg. In addition, we used EcGReg as a model to examine sixteen conserved residues in the active site and inhibitory site of the DGC domains. We propose that distal globin residues facilitate O2 binding to regulate DGC activity and BpeGReg may interact with other DGCs to form mixed signaling teams.Various GCDCs have been characterized from otovorum , 21–23. ynthesis , 8. Theyive PDEs , 8, 24. BpeGReg was created previously [Protein sequences were aligned using EBI-MAFFT , 26. Theeviously .ecGReg (GenBank accession no. NP_416007) and bpeGReg (GenBank accession no. NP_882025) genes were cloned into the pTrc99A vector (primers listed in Table A in The PfuTurbo DNA polymerase (Stratagene) and the primers listed in Tables B and C in DpnI  was then added to digest the methylated parental DNA template at 37°C for 1 hour. The mutated plasmids were transformed into E. coli TOP10 cells (Invitrogen). The coding regions of the isolated plasmids were verified by Sanger sequencing on ABI 3730xl DNA analyzer.The plasmids containing the full-length genes were used as templates for the QuickChange site-directed mutagenesis protocol (Stratagene). Briefly, BpeGReg proteins were engineered with an N-terminal hexahistidine tag by PCR. Primers are listed in Table D in For protein expression and purification, full-length and truncated http://dx.doi.org/10.17504/protocols.io.ijxccpn).The laboratory protocol of the molecular cloning has been deposited in protocols.io  at 37°C. For biofilm formation, cells were grown on LB without salt plates containing Congo red (40 μg/ml) for 40 hours at 37°C. Swimming motility was assayed on 0.3% agar plates  at 28°C for 6 hours. Due to leaky expression of the pTrc99A vector, isopropyl β-D-thiogalactopyranoside (IPTG) was not used to induce protein expression during these assays.Plasmids were transformed into BpeGReg, BpeGReg155, BpeGReg266, and BpeGReg296 were overexpressed in E. coli Rosetta2(DE3)pLysS cells for 6–8 h at room temperature. Protein expression was induced with 0.05 mM isopropyl-β-D-thiogalactopyranoside. Proteins were purified by Co2+-affinity chromatography according to Piatibratov et al. [http://dx.doi.org/10.17504/protocols.io.ikhcct6). Spectra were measured using a Cary 1E UV-Visible spectrophotometer (Varian).His-tagged ecGReg and bpeGReg encode multidomain proteins containing 460 and 475 amino acids, respectively /Lys226 (BpeGReg) for mutagenesis. To analyze the conserved residues in the DGC domain, we used EcGReg as a model and selected 16 residues in the active and inhibitory sites.In the rous ion . Our unpeviously . Here weSalmonella typhimurium ATCC 14028 to examine their in vivo DGC activities by rdar  formation and motility assays  and adhesion factors , and enhance biofilm formation [S. typhimurium develops an rdar morphotype on Congo red agar plates at 28°C but not at 37°C, indicating the expression of cellulose and curli fimbriae [EcGReg and BpeGReg [Mutated proteins were expressed in ormation . S. typhfimbriae , 32. At  BpeGReg , 30, 33.EcGReg distal residue mutants, F42A, Y43A, A68T, and M69A, produced the rdar morphotype but failed to inhibit S. typhimurium motility , this protein was also unable to induce the rdar morphotype or inhibit motility in S. typhimurium /K226A (BpeGReg), led to inactive phenotypes as well  that constitutes the middle region of the α-helix B (residues 216–228) of the middle domain [e.g. peristaltic-like shifts to extend the binding cavity of a substrate [EcGReg middle domain . All of these mutants failed in conferring the rdar morphotype or inhibiting motility in S. typhimurium  resulted in a lack of in vivo function  at the dimer interface , 36. The, or His , 40. Usiium Figs  and 4C, ion Figs  and 4C. ion Figs , while tion Figs . These rXXD motif, is found in DGC domains, including those of EcGReg and BpeGReg [365XXD368 motif of EcGReg. The D368A and D368K variants both lacked the ability to produce the rdar morphotype or inhibit motility in S. typhimurium  affected oligomerization and decreased catalytic activity, leading to the conclusion that the I-site plays a role in controlling the conformation/dynamics of DGC domains, in addition to product inhibition.An inhibitory site (I-site), consisting of an R BpeGReg . The I-s BpeGReg , 41. We ium Figs  and 4C. s et al. , who obss et al.  found thEcGReg in group 2 versus BpeGReg in group 3). The differential effects of the group 2 and 3 mutants on rdar formation and motility could be due to signaling specificity. Rdar formation and motility inhibition involve a number of processes that are regulated at multiple levels, and particular DGCs and/or PDEs are often attributed to regulating specific processes [EcGReg and BpeGReg exert their specificities are not yet clearly understood. We presume that the group 2 and 3 mutations led to decreased c-di-GMP production, resulting in threshold levels sufficient for activating one phenotype over the other. This could involve c-di-GMP receptors with different binding affinities [EcGReg, along with Ec DosP, associates with PNPase in a ribonucleoprotein complex to regulate RNA turnover [The mutations characterized in this study can be classified into three groups: 1) mutants that could no longer confer the rdar morphotype or inhibit motility; 2) mutants that could confer the rdar morphotype but not inhibit motility; and 3) mutants that could inhibit motility but not confer the rdar morphotype  activities of a few  E. coli , 44, and E. coli , our resoutcomes . Contribin vitro, which is several orders of magnitude lower than that of the full-length proteins [EcGReg (residues 173–460) exhibited four-fold less activity than the full-length protein, whereas the DGC domain alone (residues 297–460) did not have any detectable activity due to it being monomeric in solution [EcGReg and BpeGReg have DGC activity in vivo, we tested the functions of various constructs in S. typhimurium , which only contain the globin and middle domains, were able to inhibit motility , however, was not sufficient for eliciting a physiological response. Our data suggest that both the heme-based globin and middle domain are required for signaling and they may interact with other DGCs to form signaling clusters.Individual DGC domains have been shown to possess low-level enzymatic activity proteins . A consthimurium . We usedrphotype , and werrphotype . We alsomotility . CompareFig A in , indicatEcGReg and BpeGReg are required for full DGC activity. Previous studies have demonstrated that the cyclase activity of GCDCs is regulated by O2 binding to the globin sensor domain [BpeGReg constructs lacking the DGC domain resulted in motility inhibition. As these proteins did not have the ability to synthesize c-di-GMP, we postulate that the globin and middle domains of BpeGReg may be able to interact with other DGCs, PDEs, or effector proteins to coordinate c-di-GMP signaling. Thus, the function of the truncated BpeGReg proteins may be partially rescued by its partners in mixed signaling teams. There is growing evidence for the involvement of protein-protein interactions in regulating c-di-GMP networks. For example, EcGReg forms a complex with the PDE DosP [Shewanella woodyi [Pseudomonas fluorescens [Our analysis of the truncated proteins suggests that the complete structures of r domain , 21–23, PDE DosP ; the staa woodyi , 48; andorescens , 50.EcGReg and BpeGReg will provide general insights into the structure and function of GCSs. Here we provided evidence that residues Phe42, Tyr43, Ala68/Ser68 and Met69 in the distal heme pockets of EcGReg and BpeGReg could affect C-terminal DGC activity. His223/His225 and Lys224/Lys226 in the middle domains of EcGReg and BpeGReg were also required, and a number of critical residues in the A-site and I-site of the EcGReg DGC domain were identified. In addition, the full globin fold is required for GCDC activity. We further hypothesize that BpeGReg, via its globin and middle domains, may be able to form clusters with other c-di-GMP-metabolizing proteins. This may shed light on the functions of other globins, especially those single-domain proteins with unknown functions.Characterization of S1 FileEcGReg and BpeGReg in pTrc99A. Table B. Primers used for the construction of EcGReg mutants and truncated EcGReg. Table C. Primers used for the construction of BpeGReg mutants and truncated BpeGReg. Table D. Primers used for expression of truncated BpeGReg in pET-3a. Figure A. Absorption spectra of truncated BpeGReg proteins.Table A. Primers used for cloning  a) Wild-type BpeGReg (solid red line) showed heme-bound absorption spectra. b) BpeGReg155, BpeGReg266, and BpeGReg296 showed heme-bound absorption spectra, similar to that of wild-type BpeGReg.(PDF)Click here for additional data file.


The results in this study indicate that both FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys had a dual-phased dendritic microstructure; all of the phases in these alloys were solid solution phases, and no ordering was observed. Therefore, the solid solution effect significantly increased the hardness of these two alloys; in particular, FeCoNiNb alloy had the highest hardness of the alloys of interest. The corrosion resistance of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys was less than that of FeCoNi alloy because of their dual-phased dendritic microstructures. The corrosion resistance of the FeCoNiNb0.5Mo0.5 alloy exceeded that of the FeCoNiNb alloy in these solutions. However, FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys exhibited a favorable combination of corrosion resistance and hardness.This study investigates the effects of niobium and molybdenum on FeCoNi alloy, including on the microstructures and hardness of FeCoNiNb The phases of the FeCoNiNb0.5Mo0.5 alloy exceed those in the laves phase in the interdendritic region. The Nb content of the FCC1 dendrite of FeCoNiNb alloy exceeds that of the FCC2 phase in the interdendrite. The melting points of Fe, Co, Ni, Nb and Mo are 1536, 1498, 1453, 2415 and 2610 °C, respectively [0.5Mo0.5, FeCoNiNb, FeCoNi and 304SS alloys. FeCoNi alloy is very soft and has an HV of only 112, making it even softer than commercial 304 stainless steel, which has a hardness of HV 185. However, adding Nb/Mo to FeCoNi alloy significantly increased its hardness. FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys had higher hardness values of HV 629 and HV 798, respectively. The microhardness values of the FCC-dendrite and eutectic-interdendrite of the FeCoNiNb0.5Mo0.5 alloy were HV 677 and HV 317, respectively. The microhardness values of the FCC1-dendrite and eutectic-interdendrite of FeCoNiNb alloy were HV 840 and HV 233, respectively, to which the solid solution effect contributed. The atomic radii of Fe, Co, Ni, Nb and Mo are 0.124, 0.125, 0.125, 0.143 and 0.140 nm, respectively [0.5Mo0.5 and FeCoNiNb alloys were solid solution phases; a large lattice distortion was thus expected due to the presence of elements with larger radii, Nb and Mo. Therefore, the solid solution effect was the cause of the increased hardness.0.5Mo0.5, FeCoNiNb, FeCoNi and 304SS alloys in 1 M deaerated H2SO4 solution and indicates important characteristics, such as corrosion potential (Ecorr), passivation potential , critical current density of the anodic peak (icrit), passive current density (ipass) and breakdown potential (Eb). icorr) associated with each polarization curve was obtained from the intersection of the free corrosion potential and the cathodic Tafel line. The corrosion current densities of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys exceeded the icorr of the single FCC-phased FeCoNi alloy, because they both had a dual-phased microstructure and thus easily formed local cells between the anodic and cathodic regions. However, the corrosion current densities of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys were smaller than the icorr of 304 stainless steel. Therefore, the corrosion rates of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys were smaller than that of 304 stainless steel in 1 M deaerated H2SO4 solution at 30 °C. The corrosion potentials of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys were only slightly lower than the Ecorr of FeCoNi alloy, but these alloys were still nobler than 304 stainless steel. However, the passive current densities of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys exceed those of 304 stainless steel and FeCoNi alloy.0.5Mo0.5 and FeCoNiNb alloys after the polarization test in 1 M deaerated H2SO4 solution at 30 °C. The dendrites of FeCoNiNb0.5Mo0.5 alloy were an FCC phase and the interdendrites were a eutectic structure of FCC and laves phases. The laves phase was the matrix of the interdendrite, which was corroded during the polarization test in 1 M deaerated H2SO4 solution at 30 °C, as shown in 0.5Mo0.5 alloy was almost unchanged after the polarization test. 2SO4 solution at 30 °C. The dendrites of FeCoNiNb alloy were an FCC1 phase, and the interdendrites were a eutectic of FCC1 and FCC2 phases. The FCC2 phase in the interdendrite of FeCoNiNb alloy was the main corroded phase in the polarization test in 1 M deaerated H2SO4 solution at 30 °C. The FCC1 phase of FeCoNiNb alloy was only slightly corroded after the polarization test. Therefore, the main corroded areas of these two alloys are the interdendrites. Since the interdendrite area of FeCoNiNb alloy was less than that of FeCoNiNb0.5Mo0.5 alloy, the corrosion current density of FeCoNiNb alloy was less than that of FeCoNiNb0.5Mo0.5 alloy. 0.5Mo0.5, FeCoNiNb, FeCoNi and 304SS alloys in 1 M deaerated NaCl solution. 0.5Mo0.5 and FeCoNiNb alloys were close to the Ecorr of FeCoNi alloy, these alloys were all nobler than 304 stainless steel. This result indicates that adding Nb/Mo to FeCoNi alloy did not significantly change its Ecorr, although but did change its microstructures to dual-phased ones. However, the dual-phased microstructures of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys significantly influenced on the icorr of the alloys. The corrosion current densities of FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys exceeded the icorr of FeCoNi alloy; but smaller than the icorr of 304 stainless steel. FeCoNiNb0.5Mo0.5 alloy had a large passivation region because of the addition of molybdenum. Adding Mo can reportedly increase the corrosion resistance of the alloy in a solution that contains chloride ions [0.5Mo0.5 alloy. The cathodic limiting current densities (iL) in FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys were observed. The cathodic limiting current density corresponds to the maximum reaction rate, which is herein limited by the diffusion rate of hydroxyl ions (OH−) in solution [ide ions  because solution . Howeversolution .0.5Mo0.5 and FeCoNiNb alloys after the polarization test in 1 M deaerated NaCl solution at 30 °C. As for the FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys that were tested in 1 M deaerated H2SO4 solution, their main corroded areas were the interdendrites. 0.5Mo0.5 alloy after the polarization test. The corroded phase was the laves phase in the interdendrite of FeCoNiNb0.5Mo0.5 alloy. The FCC-structured dendrite and FCC phase in the interdendrite were not corroded and so their original morphologies were retained. 0.5Mo0.5 and FeCoNiNb alloys had dual-phased dendritic structures. The phases in FeCoNiNb0.5Mo0.5 alloy were FCC and laves phases, and the dendrites exhibited an FCC phase. The phases in FeCoNiNb alloy were FCC1 and FCC2 phases, and the dendrites exhibited an FCC1 phase.Both FeCoNiNb0.5Mo0.5 alloy; and both FeCoNiNb0.5Mo0.5 and FeCoNiNb alloys were harder than FeCoNi alloy and 304 stainless steel.The hardness of FeCoNiNb alloy was higher than that FeCoNiNb0.5Mo0.5 alloy exceeded that of FeCoNiNb alloy in 1 M H2SO4 and 1 M NaCl solutions. The corrosion resistance of these two alloys was less than that of FeCoNi alloy but greater than that of 304 stainless steel in these two solutions.The corrosion resistance of FeCoNiNb


Furthermore, the compressive strength of cement pastes at the age of 28 days increased by 37.9% than that of the plain sample. Improvement of performance of cement-based materials can be partly attributed to the refinement of the pore structure. In addition, AFM was employed to characterize the nanoplatelet thickness of CCNPs and the pore structures of the cement-based composites were analyzed by MIP, respectively. CCNPs composite cement best performance could lay the foundation for further study of the durability of cement-based materials and the application of decontaminated seashells.Nanomaterials have been widely used in cement-based materials. Graphene has excellent properties for improving the durability of cement-based materials. Given its high production budget, it has limited its wide potential for application in the field of engineering. Hence, it is very meaningful to obtain low cost nanoplatelets from natural materials that can replace graphene nanoplatelets (GNPs) The purpose of this paper is to improve the resistance to chloride ion penetration by optimizing the pore structure of cement-based materials, and another point is to reduce investment costs. The results illustrated that low cost CaCO The progress of nanotechnology has brought new insights to improve the properties of cement-based materials with the aid of nanomaterials in recent years ,2,3. 2) [2O3) [2) [Nanomaterials  are divi2) ,6,7,8,9,2) [2O3) ,12,13,142O3) [2) ,16,17,182O3) [2) ,24,25,262O3) [2) ,31,32,33The durability exists an important performance of cement-based material. In the field of marine engineering, there are many factors that affect the durability of cement-based materials, including alkali-aggregate reaction, acid erosion, alkali erosion, and chloride ion penetration etc. Yet, the penetration of chloride ions will cause serious and irreversible damage to cement-based materials. Therefore, it is of great significance to improve the impermeability of cement-based materials.GNPs is a kind of nanoscale materials consisting single-layer or multi-layer graphene flakes ranging in thickness from 1 to 100 nm. Compton  studied Seashells are heavily abandoned in coastal areas ,40,41, wIn the paper, low cost CCNPs were firstly obtained under alkali treatment of seashells powder. Moreover, the effect of CCNPs incorporated on the performance of cement-based materials was observed. The nanoscale thickness of CCNPs was characterized by atomic force microscope (AFM) images. Besides, the pore structure of cement composites was characterized by mercury intrusion porosimetries (MIP) and the mechanism of the introduction of CCNPs was revealed. The best performance of CCNP composite cement lays the foundation for further study of the durability of cement-based materials and the application of decontaminated seashells.Type I Portland Cement (CEM I 42.5) plays the role of the main binder, which was provided by Qufu China United Cement Co., Ltd. . Mussel shells were collected from Lianyungang City, Jiangsu province, which were arbitrarily abandoned in the seafood market. Sodium hydroxide (AR) and ethanol solution (AR) were purchased from Xilong Reagent .Mussel seashells were rinsed and dried in an oven at 65 ± 5 °C for 24 h, followed by cracking and grounding into powders to pass 75 μm sieve by using a vibration crusher. Sodium hydroxide solution with a mass fraction of 5% was used as the reaction solvent, accompanied by water-curing treatment at 80 °C with maintaining magnetic stirring for 7 h, then ultrasonic dispersion for 1.5 h and centrifugal cleaning solution to neutral, finally the powder was dried in an oven at 65 °C.Cement paste was mixed with water-cement at mass ratio of 0.28, which meets the fluidity of cement paste under the standard requirements. 0.01, 0.02, 0.03, and 0.04% of CCNPs by weight of cement were used in this research.Cement mortar was mixed with cement to sand at mass ratio of 1:3. The water to cement ratio of 0.5. 0.01, 0.02, 0.03, 0.04, 0.07, and 0.1% CCNPs by weight of cement were used in this study.The chemical composition of mussel seashells powder was determined by X-ray fluorescence spectrometry . In addition, the particle size distribution of the mussel shell powder was measured by using a laser particle size analyzer . Rigaku D/Max-2500 XRD, Cu-Kα (1.541874 Å) was used to investigate the phase structure of the prepared samples.The physical properties of CCNPs were tested by the following characterization: particle morphology disclosed through a scanning electron microscope ; and the thickness of CCNPs were considered by atomic force microscope .The compressive strength of cement pastes was examined at various ages of 3, 7, 28 and 56 days using 2 cm cube specimens. A mean value of six paste cubes was used for the compressive strength test. The compressive strength of cement mortar was examined at various ages of 3, 7, 28 days by testing 4 cm cube specimens. A mean value of six mortar cubes was used for the compressive strength test.NT build 492  (RCM met−1), and 10 wt % NaCl solution. Besides, because of the apparent diffusion coefficient of chloride ion in cement-based materials, the other half of the specimen was spurted with AgNO3 solution to disclose the chloride penetration depth, which can be quickly and accurately measured by silver nitrate colorimetric method.In this study, specimens were obtained from Φ110 × 220 mm cement mortar cylinders. The migration cell is full of 0.1 M/L NaOH . L is the thickness of the specimen, accurately to 0.1 mm. T is the mean value of the initial temperature and the end temperature of the anodic solution. Xd is the mean value of the penetration depth of the chloride ion, accurately to 0.1 mm. The half of the specimen was measured for 10 values. T is the duration time of the test.The calculation of the chloride penetration value from migration test was obtained with the simplified Equation (1) (1)DRCM=It can be seen from the 3 phases (approximately 96.5%) and minor mineral composition. The mineral phase of calcium carbonate is demonstrated to be calcite or aragonite [3.The chemical composition of mussel seashells was given in ragonite ,41,43. IXRD was used to investigate the phase structure of the prepared samples. Mussel shells have traditional ‘brick and mortar’ arrangement on the fractured surfaces of the mussel shells , which iHigh magnification AFM was utilized to analyses the thickness and the finer structure of CCNPs, which was presented in The porosity and pore distribution are two main factors affecting the durability and strength of cement-based materials. Cementitious materials with lower porosity and finer pores show better durability and higher strength. The effect of the incorporation of CCNPs on the porosity and pore size distribution was determined by MIP is discussed below.It can also be observed that with the 0.04% of CCNPs incorporating to cement mortar, the result shows that both the less harm pore and large pore increase while the harmless pore decreases compared with the control sample. That is because when CCNPs are introduced into the cement, they will be surrounded by the hydration product of the cement and remain in it. When chloride ions intrude into cement, the permeation path of chloride ion can be changed and extended, thus improving its impermeability .However, with the increasing content of CCNPs, the properties of cement-based materials decrease even lower than those of control samples. It could be concluded that, due to the effect of CCNPs stacking effect and its irregular shapes, coupled with the combined effect of weakening the nanoscale effect, when the amount of CCNPs increases, more water was needed for the dispersion of CCNPs, which easily results in an aggregation effect in the mix. As reported ,43,44,45The result of the chloride penetration impermeability of cement mortar specimens is shown in As the curing age extended, the compressive strength increased correspondingly, because the process of the cement hydration had gradually accomplished. Seashell landfill brings about a great deal of pollution and waste into the environment. Recycling waste seashells can effectively solve the problems. In this study, Nanoscale CCNPs were obtained from alkali-treated mussel shells powder. With incorporating CCNPs to the cement-based materials at lower dosage, the results indicate the positive effect on the resistance of chloride ion penetration and the compressive strength. Besides, it can bring considerable economic and environmental efficiency. Therefore, the following conclusions can be made:Nanoscale CCNPs can be successfully obtained from mussel shell powder by alkali treatment. Specific test process: the mussel shell powder was placed in the mass fraction of 5% sodium hydroxide solution, 80 °C water bath heating, and supplemented by 1.5 h of ultrasound correspondingly.CCNPs have an efficacious influence on chloride ion impermeability and compressive strength by refinement of the pore structure of cement-based materials. The cement mortar incorporating 0.04% CCNPs resulted in a 16.1% reduction in chloride penetration as compared with the control mortar. Also, the cement paste including 0.01% CCNPs provided 37.9% higher compressive strength than the control paste. Yet, CCNPs should remain in the low dosage because of the agglomeration effect.


This study explores the drivers of site selection amongst livestock owners under conditions of increasing animal numbers following a low point in the 1990s. Our major goal was to understand whether livestock owners are acting as ‘optimal foragers,’ targeting areas of highest forage availability as they colonise previously empty areas. The results presented here suggest that they do not. Initially, distance from home settlement was the dominant determinant of site occupancy, with closer sites occupied earlier regardless of other characteristics. Some owners remained on depleted vegetation for longer than would be predicted under conditions of optimal foraging, indicating that distance-related costs constrained resource matching. In the latter period, increases in livestock wealth encouraged the occupation of distant sites exhibiting higher vegetation density and water quality, but some owners still occupied highly depleted sites. Improved transport and water supply infrastructure are needed if pastoralists are to optimise resource use across the landscape.The online version of this article (doi:10.1007/s10745-016-9870-5) contains supplementary material, which is available to authorized users. Whilst et al.et al.Our study area is one of the most important winter pastures in Kazakhstan and access brings significant economic benefits through a reduction in winter feed costs  . They will leave a patch when the marginal intake rate drops to the average for the habitat. This type of  Charnov : The ricpopulation level  for a richer patch where higher initial resource density compensates for the time travelled.This theory helps us to investigate resource depletion and the potential for pasture degradation, because resource density metrics  are often easier to measure than forage intake by animals. For this reason, giving up density (GUD), the resource density at which animals leave a patch, is often measured in studies of MVT as an indicator of the intake rate at which patches are left Brown . For thiet al.et al.et al.et al.et al.Key MVT predictions have been validated in domestic animals under controlled conditions  of the better forage in distant patches. Once MOC become larger than distance-related costs, it is worth changing patches. Herd size is likely to offset distance by reducing costs through economies of scale. We use this model to set up our hypotheses.(i)Resource density may be higher at occupied sites than unoccupied sites if herders are targeting higher vegetation density under optimal foraging conditions and have not yet depleted patches to the landscape average.(ii)Considering the expanding nature of site selection over time and ignoring the cost of travel, we predict that sites occupied earlier have a higher resource density than those occupied later, or at least this would have been true during the early stages of re-occupation.The non-equilibrium situation means that there is unlikely to be a direct relationship between resource density and stocking rate, but the situation of expansion across a single zone is suitable to test the following two hypotheses, which relate to the beginning of the expansion process and would hold if gain is strongly correlated with resource density and if travel-related costs are low:(iii)Higher levels of relative depletion (not absolute resource density) outwards from wells at occupied sites than unoccupied sites;(iv)Lower depletion levels at more recently occupied sites than sites occupied for several years or occupied and then abandoned;(v)An overriding role for distance in the system: sites close to our putative central point, the village, would be colonized first regardless of forage availability considerations;(vi)Amongst occupied sites and over time we would expect to see a positive relationship between vegetation density and distance from villages, as the ratio of gain to cost should start to equalise over the landscape. We would also find negative relationships between forage depletion and distance as forage is depleted closer to villages;(vii)Lastly, if herd sizes have a mitigating effect on costs, we would expect to see a positive relationship between herd size and distance from the central point.If travel costs are much higher than the MOC of not moving, then livestock will deplete resources to far below the average level for the landscape, leading to very low GUDs. In such cases, we would expect to see that hypotheses (i) and (ii) would be refuted. In addition we would expect to observe the following conditions:et al.These hypotheses may be tested straightforwardly in an environment in which patch quality (before grazing) is distributed randomly. Yet patch quality distribution is not random, but exhibits gradients across the landscape. Moreover, forage density is certainly not the only site characteristic of possible interest to herders. Water quality  and accessibility (depth) have been shown to influence site selection, whilst snow cover and relief affect vegetation and site accessibility , Chu, Moiynkum and Baital, whose populations were concentrated in the settlements of Ulan bel’, Malye Kamkale, and Sary Ozek respectively  over 12 month periods starting from October 2002 and October 2011. Overall, 118 unique livestock owners were recorded. Of these, full livestock ownership information was available for 80 owners in 2003 and 84 in 2012 (58 appeared in both years). According to the census data, 49 owners used the Moiynkum desert at some period of the year in 2002-2003, and 30 in 2011–2012 . The information on stock type, numbers, water source, and monthly location name were entered into a database. Numbers were converted to standard livestock units (LU) often used by pasture scientists in Central Asia and similar to cattle-sheep ratios used on rangelands elsewhere  during five fieldwork periods between 2012 and 2014. Interviewees included livestock owners, shepherds, village mayors, veterinary officers, former employees of the state livestock farm, and employees of the state forestry department. Interviews were used to gather livestock owners’ perceptions on site selection and to identify important variables affecting decision-making. The results are discussed in detail elsewhere , whilst density (stocking rate) information was available on a monthly basis for two single year periods running from the months of October 2002 and 2011.Concerning the exact locations of used sites, many of the surveys and interviews were conducted at grazing sites, recorded by GPS. Livestock owners and other knowledgeable interviewees identified additional used locations on a set of 1:50,000 Soviet era topographical maps. The majority of these sites were then visited by vehicle with key informants and recorded by GPS. The locations of those sites that could not be visited were digitised directly from the georeferenced topographical maps on which all wells are shown, many with names. Most such locations were verified on the maps by more than one interviewee. All locations, with their coordinates, were inputted into a relational database, thus providing exact locations for every site listed in the livestock survey table.Livestock presences were recorded as locations having any stock presence over the year, whilst absences were defined as locations having wells that existed in the Soviet era, but which were not recorded as occupied either in 2002–3, or between 2011 and 2014. These may be considered potential sites, and were digitised from the topographic maps .sovkhoz boundaries. For this reason the maximum extent of our analysis was set to cover an area stretching up to 30 km from these boundaries. Within this area, of the total of 445 occupied and potential sites, 282 had infrastructure  and were considered to be major sites likely to have been used as bases in the past; others consisted of isolated wells and may have been occupied for shorter periods on a temporary basis. Both sets of sites were used in analysis for comparison, but the set of sites with infrastructure was considered to be the most conservative estimate of potential livestock locations existing during the later Soviet period . Twenty-seven of these were recorded with full livestock number information for our specific census period October 2011–October 2012. The rest were recorded as having livestock presence at some point between October 2011 and April 2014. These data represent two snapshots of well occupation in a complex 20 year-long process of well occupation and abandonment. Our data do not include information on the exact dates and length of occupation periods between the two study periods. However, the year of first occupation since the collapse of the sovkhoz was obtained for a subset of 29 wells for more detailed investigation of site sequencing. The sets of occupied sites are different in the two time periods, altering the choice of unoccupied sites. This is addressed by explicitly comparing site characteristics between sites occupied in 2011–2014 alone, occupied in 2003 and then abandoned, and occupied in both periods.Overall, livestock were present at 26 locations in 2002/2003 and 40 during the full fieldwork period of 2011–2014  is used worldwide as an indicator of green biomass . Relief, in the form of sand dunes, was mapped at a 30 m resolution using visual enhancement and interpretation of an ASTER Digital Elevation Model (DEM).Relationships between distance from settlement and NDVI, date of first occupation and NDVI, stocking rate and NDVI gradient, and well depth and first date of occupation were explored by ordinary least squares linear regression. Differences in mean NDVI, NDVI gradient, distance from settlement, well depth and snow cover between locations with stock presence and absence were assessed using Welch’s t-test; differences in these variables between the four site stocking categories  were assessed using one-way ANOVA. All statistical analyses were carried out in R  so the area was essentially ‘full up’ with potential sites in Soviet times. The distribution of wells (with infrastructure) mapped within all sovkhoz boundaries is close to a distributed regular pattern  and within Chu sub-district alone follows a highly distributed pattern . The equivalent analysis using all wells reveals a random pattern, suggesting that a planned effort was made to regularly space sites with infrastructure and confirming that the presence of infrastructure might be the best metric to define absences . These findings also suggest that either underlying environmental factors did not influence well distribution, or that these factors do not display a spatial gradient or clustering pattern.Wells with infrastructure in the sand desert portion of our three Today, large areas of the Moiynkum desert are unoccupied but the number of occupied sites has risen from 26 in 2002–3 to 40 in 2012–13, with 15 sites occupied in both periods. Some sites are occupied all year but others only seasonally. Seasonal use is now highly variable, ranging from one to four seasons rather than in spring/autumn (north of the desert) and winter (southern desert) which characterised the Soviet period .sovkhoz Moiynkum, which are unoccupied. We discuss how forage resources, and other natural factors possibly influencing site selection, vary across the desert.The occupation of new sites has occurred mostly to the south and east Fig. a. There Artemisia and Haloxylon spp. . This pasture makes poor grazing outside the autumn and only two occupied wells were located here. Further south, the soil becomes progressively sandier  give way to vegetation dominated by shrubs such as Calligonum, Haloxylon, Astragalus spp. which, together with an understory including Ceratoides, Kochia and Atriplex spp., provide good forage all year round. By far the majority of wells are located in this vegetation type on sandy soils .Our sample of wells, representing those for which respondents had knowledge, exhibited decreasing salinity towards the south from settlements, a factor identified as important in site selection by herders . But at a radius of 1 km, where grazing pressure is greatest, occupied sites had significantly lower NDVI . The 2003 set of occupied sites exhibits very strong differences in NDVI when compared with sites unoccupied at that time, even at 6 km radius from wells, with occupied sites having significantly lower NDVI . These differences were also noted using NDVI in the year of use instead of 12 year annual average NDVI. Thus site selection decisions made in individual years are unlikely to depend on resource density characteristics in that specific year, and resource densities in a given year do not differ radically from long term averages.Our first hypothesis was that occupied sites may be those with higher long term average resource density. We examined this by comparing average annual NDVI (2001–2012) at occupied and unoccupied wells. Taking livestock occupancy during the 2011–2014 period, there was no significant difference in mean NDVI values between the 37 occupied and 93 unoccupied locations within the three former sovkhoz. Whilst no relationships were evident at 6 km radius, a weak relationship did emerge closer to wells  when the full set of sites with wells (not only those having infrastructure) are included in the analysis. Whilst wells with infrastructure are likely to have been occupied in the past, those without infrastructure may have been little or never occupied, thus amplifying the potential difference in accumulated grazing pressure between occupied and unoccupied sites. This also suggests that past grazing effects are detectable for long periods.However, we have seen that there is also a north-south gradient in ‘natural’ vegetation density related to climatic factors rather than livestock grazing. Our most recently occupied sites, as well as many unoccupied sites, appear to be located further south. Thus we must examine not only absolute resource density, but relative depletion levels around wells in order to better understand the process behind the observed patterns.sovkhoz boundaries, sites occupied only in recent times  exhibited no discernible difference in NDVI gradient in 2012 from unoccupied sites, which, as would be expected, have a median gradient of around zero  and the extent or size of the gradient in either 2003 or 2012. This may be because stocking rates are not variable enough to cause detectable differences in depletion level.It should be noted that although we have found relationships between livestock presence and resource depletion, relationships were not found between actual livestock negative, as those sites occupied first are furthest north, where precipitation is lowest. Those sites occupied in 2003, but abandoned before the subsequent study period, have low NDVI gradients (little depletion) in 2012  concerning distance from the home settlement  alone, as so few stock from Sary Ozek are located outside the settlement. In this sub-district most livestock are coming from the settlement of Ulan bel’. Results confirm that indeed occupied sites were significantly closer to Ulan bel’ than unoccupied sites in 2003 . This is because there are many unoccupied sites in the highly vegetated south of the sandy massif. These sites are potentially available and indeed a few were occupied by 2013.Yet the impact of distance on site selection still had consequences during the later study period. If we compare sites occupied in 2011–14 with the theoretical larger universe of unoccupied sites across the wider Moiynkum zone, then NDVI values at sites occupied in 2011–14 are sovkhoz boundaries alone that there is most likely to be equalising of cost-gain ratio. We did indeed find a strong positive relationship between distance from the home settlement and NDVI at sites that had been occupied at any time during the study period ; R2 = 0.76, p < 0.0001). However, there is also a strong relationship between NDVI and distance for sites that were not occupied in either period ; R2 = 0.57; p < 0.0001), albeit with a lower slope. We would also expect to find negative relationships between forage depletion and distance from settlement at occupied sites, as forage is depleted closer to settlements . Here although a slightly negative relationship was observed, it is extremely weak (b1 = −9.6e-05 (se = 4.5e-05); R2 = 0.08, p < 0.05).If costs increase with distance then we would expect GUD to be lower closer to settlements and higher farther away, as the ratio of cost to gain starts to equalise over the landscape (hypothesis vi). By the end of our study period, we would thus expect to see a gradient of resource density outwards from settlements at sites occupied during our study period, but not at unoccupied sites. However, this hypothesis cannot be confirmed due to the natural north-south increase in resource density evident at both occupied and unoccupied sites Fig. c. It is n = 27). The difference in herd size between sites occupied in 2012 and sites occupied in both 2003 and 2012 was also non-significant.Lastly, we expected that if economies of scale could mitigate travel costs, herd size should be positively related to distance from the village. We explored the relationship between both average herd size over the months each site was occupied, and annualised stocking rate (in LUY) and distance from village for the year 2012. No relationships were apparent here for either variable, however the sample size of sites for which stocking information was available was small , depth and type of the well (which may affect extraction cost), topography (which may affect travel cost), and snow cover (which may impede grazing in winter but provide important moisture in spring) . The possible conclusion that shallower wells tend to be occupied later on is supported by weak relationships between well depth and both date of first occupation and use period category  for which depth data were available was small. In 2003, occupied wells were significantly deeper . There are very few data for wells unoccupied in either period. This is because herders are unsure about the status of wells that have not been visited for a long time. However, interviews do confirm that pumping costs increase with well depth and that water quality is an important pull factor in site selection (Kerven n = 58). Sites having only bore holes may be the least likely to be occupied today as they require the most powerful pumps. Indeed, of the 25 sites at which bore holes alone are indicated, only five were occupied in either 2003 or 2011–14 (of which three have been occupied since 2012). Of the 33 sites with shaft wells or both types, 18 (54%) were occupied . Most unoccupied sites lie directly south of Ulan bel’ nearer to the metalled road. The fact that most of them are bore holes may explain why those sites, despite being easily accessed, are rarely occupied , winter is still a key grazing period. Snow cover as measured by satellite imagery is significantly lower at more recently occupied sites (2011–14 only) compared to those occupied in 2003 alone, but the absolute mean differences are very small . Moreover, more snow may be positive for spring growth, thus in interviews some herders considered snow to be an important factor determining site selection whilst others did not, depending on season of use. This factor is also likely to be confounded with geographical location, which as we have seen, correlates with other factors varying along the same north-south gradient.To summarise, it appears that over time, herders have been increasingly targeting shallower and less saline wells in areas with higher vegetation density.A major goal of this paper was to understand whether livestock owners are acting as ‘optimal foragers,’ targeting areas of highest forage availability as they colonise previously empty areas. The results presented here suggest that they do not follow the classical MVT  against the constraints imposed by distance. The relative outcome of these two opposing forces is measured in our specific factor of interest – resource depletion, interpreted in the theory through the concept of GUD. This approach has allowed us to show empirically over a large area what range scientists in Kazakhstan have observed since independence – an uneven use of rangeland resources leading to a combination of localised overuse and abandonment


Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon’s N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.Lon is an essential, multitasking AAA Interestingly, the BsLon AP-domain crystallized as an open, helically arranged hexamer when bound to six molecules of ADP, but the MtaLon’s AP-domain adopted a planar conformation. Unfortunately, electron microscopy (EM) studies of Lon and its bacterial and yeast counterparts FtsH and m-AAA proteases have likewise failed to provide a clear picture of the overall organization of the protease16171828293031Therefore, the Lon protease is a dynamic protein whose functional domains  are present on a single polypeptide chainhLon S855A mutant, which retains near wild-type levels of ATPase activity. We determined the structure of this mutant after its incubation with the non-hydrolyzable ATP-analogue AMP-PNP and with ADP at resolutions of 15 Å and 21 Å, respectively. In order to study the role of hLon’s N-terminal domain, we also determined the enzymatic properties of a hLon protein lacking its first 270 amino acids (hLonΔ270) and acquired insight into its structure by a cryo-EM analysis. The deletion includes the 114 amino acids of the mitochondrial targeting pre-sequence and 156 amino acids from the N-terminal domain  containing the proteolytic and the ATP-binding domains (AP-domain). The “head” therefore contains the catalytic chamber of the protein complex, which has a 20 Å diameter opening at its C-terminal side, but no opening at its N-terminal side . However, the open-ring helical arrangement contrasts to the closed-ring planar arrangement of MtaLon’s AP-domain with three ADPs and three nucleotide-free monomers, which may be a consequence of either a different occupancy of ADP-binding sites or restrictions imposed by crystallization. Considering the planar arrangement of the AMP-PNP incubated hLon structure, it is likely that the lock-washer form of hLon’s AP-domain is a true consequence of ADP binding to a full-length hLon hexamer in physiological conditions. The manifold arrangements of the existing structures of Lon’s AP-domain hexamer document its flexibility, required for its allosteric operations. Lock-washer conformations were also observed in the cryo-EM structures of the D1 ring of the NSF ATPase and in the 26S proteasome42Compared to the hLon mutant lacking the first 270 residues, i.e. missing the 114 amino acids of the mitochondrial targeting pre-sequence and the first 156 amino acids of the mature protein. Secondary structure prediction algorithms of hLon’s N-terminal domain indicated that it could be divided into two subdomains separated by an unstructured regionhLon still forms a multimeric complex  was prepared by in-fusion cloning into a pOPINJ vectorExpression, purification, and Lon_271_FW: AGTTCTGTTTCAGGGTACCATGGTGGAGGTAGAGAACGTTGTCLon_960_RV: CTGGTCTAGAAAGCTTTCACCGTTCCACGGCCAGhLonThe construct was verified by DNA sequencing (Macrogen). Protein expression and purification was performed following the protocol for wt 2, 10% (v/v) glycerol). The flow rate was 0.4 ml/min. The peak fractions were analyzed by SDS-PAGE, concentrated on Microsep Advance 100 K columns  and stored at −75 °C. The concentration of protein was determined using the BCA kit .Analytical gel filtration was performed with a Superose 6 10/300 GL  column using buffer A , concentrated to 1 mg/ml, were diluted 5× in buffer B  and 3 μl of the dilution were applied to freshly glow-discharged Quantifoil 2/2 grids . The grids were vitrified by plunge-freezing into liquid ethane in a Vitrobot machine  after 2 s blotting (blotting force 2) at 4 °C and 100% humidity.All three specimens  at an operating voltage of 100 kV with a FEI Falcon I 4 k × 4 K direct detection camera at IGBMC Strasbourg, France. Since the overall organization of the Lon protease has been intriguing so far, we made use of the markedly better performance of the Falcon I DED at voltages lower than 200 kV in the low-resolution range of 0–~0.25 × Nyquist frequency2. In total, 1074 images of ADP-incubated hLon S855A and 575 images of AMP-PNP-incubated hLon S855A were recorded.Datasets of both hLonΔ270 dataset was recorded with a Cs-corrected FEI Titan Krios TEM at an accelerating voltage of 300 kV at IGBMC Strasbourg, France, on a Falcon II 4 K × 4 K direct-detection camera operated in movie mode. The nominal acquisition magnification was 47000×, resulting in a final image pixel size of 1.42 Å. Seven of the recorded frames (frames 2–8) covering an accumulated electron dose of ~20 electrons/Å2 were saved for further processing. The EPU-driven image underfocus varied between −1.3 μm and −2.4 μm; in total 2980 stacks of 7 frames were recorded.The hLon S855A, 458 images of the AMP-PNP incubated hLon S855A, and 2739 images of the hLonΔ270 specimens were selected for further processing. The hLonΔ270 frames were aligned and summed with the GPU-based dosefgpu_driftcorrhLonΔ270 specimen. The value of the Cs coefficient of spherical aberration was set to 0.1 in the processing of the hLonΔ270 dataset. Particles were picked semi-automatically using the e2boxer program from EMAN2hLon S855A ADP-incubated particles, 97500 hLon S855A AMP-PNP-incubated particles, and 39800 hLonΔ270 particles were extracted. The box size was set to 224 pixels for the hLon S855A samples, and to 192 pixels for the hLonΔ270 specimen.After CTF and ice quality inspection, 498 images of the ADP-incubated hLon S855A AMP-PNP-incubated particles, 32500 hLon S855A ADP-incubated particles, and 12450 hLonΔ270 particles were selected for subsequent 3D classification. Inspection of the resulting 2D class averages of full-length specimens revealed top, side, and tilted views .In order to create an initial model for the 3D classification of both full-length In Relion, these initial 3D models were again low-pass filtered to 120 Å and subjected to grey-scale invariant cross-correlation in the first iteration of the 3D classifications. All 3D classification runs were performed with the initial starting model, and the number of classes in each 3D classification run was selected so that at least one “empty”, i.e. very sparsely populated class, resulted.hLon S855A AMP-PNP-incubated dataset was performed with 5 classes; 30000 particles from the most populated class were then passed through another classification run with 3 classes. From this 2nd run, 23170 particles were selected for final 3D refinement. 3D classification of the hLon S855A ADP-incubated dataset was performed in 3 runs with 6, 3, and 6 classes, respectively. After the 3rd run, 10809 particles were available for 3D refinement. In each 3D classification run on the full-length Lon datasets, only those particles contributing to classes with well-formed N-terminal domains were selected for further 3D processing.The first 3D classification of the hLon S855A–AMP-PNP structure, and 22.4 Å hLon S855A–ADP structure. The reported resolutions were estimated using Relion’s gold-standard Fourier Shell Correlation at the level of 0.143. Both full-length hLon S855A structures were then masked, resulting in a resolution of 15 Å for the AMP-PNP-incubated structure and 21 Å for the ADP-incubated structure. Local resolution maps were computed using ResMapB. subtilis Lon hexamerThe 3D maps of the major classes acquired from the final 3D classification run were low-pass filtered to 40 Å and used as initial models for 3D refinement, giving an 18.3 Å resolution hLon S855A AMP-PNP-incubated particles against a synthetic soft-edged cylinder with a length of 220 Å and a diameter of 140 Å. The resulting structure was identical to the one showed in In order to check the influence of the starting model on the resulting quaternary structure of the full-length protein, we processed the 23170 B. subtilis Lon AP-domain (PDB ID 3M6A)E. coli Lon N-terminal domain crystal structure (PDB ID: 3LJC)Rigid-body fitting of the AP-domains of the reconstructed structures was performed by the computation of cross-correlation coefficients between the reconstructed cryo-EM maps and chain A of the hLon S855A, both reconstructed cryo-EM maps were loaded into Chimera, aligned, and fitted with the six corresponding B. subtilis AP-domain monomers from the Sculptor docking. The positions of the ADP molecules in the hLon S855A AMP-PNP Lon reconstruction could be approximated by a virtual plane, which served as a reference also for the measurement of both vertical and angular distances between the ADP molecules in the hLon S855A ADP Lon reconstruction. Distances in the cryo-EM maps were measured using ImageJMeasurements in the fitted PDB structures were performed in UCSF Chimera. In order to compare the two nucleotide-state structures of Accession codes: The reconstructed density maps of the cryo-EM hLon S855A incubated with AMP-PNP and ADP have been deposited in the Electron Microscopy Data Bank under accession codes EMD-3275 and EMD-3274.How to cite this article: Kereïche, S. et al. The N-terminal domains plays a crucial role in the structure of a full-length human mitochondrial Lon protease. Sci. Rep.6, 33631; doi: 10.1038/srep33631 (2016).


Motivation: Over the last decades, vast numbers of sequences were deposited in public databases. Bioinformatics tools allow homology and consequently functional inference for these sequences. New profile-based homology search tools have been introduced, allowing reliable detection of remote homologs, but have not been systematically benchmarked. To provide such a comparison, which can guide bioinformatics workflows, we extend and apply our previously developed benchmark approach to evaluate the ‘next generation’ of profile-based approaches, including CS-BLAST, HHSEARCH and PHMMER, in comparison with the non-profile based search tools NCBI-BLAST, USEARCH, UBLAST and FASTA.Method: We generated challenging benchmark datasets based on protein domain architectures within either the PFAM + Clan, SCOP/Superfamily or CATH/Gene3D domain definition schemes. From each dataset, homologous and non-homologous protein pairs were aligned using each tool, and standard performance metrics calculated. We further measured congruence of domain architecture assignments in the three domain databases.Results: CSBLAST and PHMMER had overall highest accuracy. FASTA, UBLAST and USEARCH showed large trade-offs of accuracy for speed optimization.Conclusion: Profile methods are superior at inferring remote homologs but the difference in accuracy between methods is relatively small. PHMMER and CSBLAST stand out with the highest accuracy, yet still at a reasonable computational cost. Additionally, we show that less than 0.1% of Swiss-Prot protein pairs considered homologous by one database are considered non-homologous by another, implying that these classifications represent equivalent underlying biological phenomena, differing mostly in coverage and granularity.Availability and Implementation: Benchmark datasets and all scripts are placed at (http://sonnhammer.org/download/Homology_benchmark).Contact:forslund@embl.deSupplementary information: Supplementary data are available at Bioinformatics online. Modern molecular biology relies on evolutionary conservation of properties between entities such as genes and proteins that are homologous, i.e. share descent from a common ancestor. As a historical property homology is unobservable but can be inferred from statistically significant similarity under the proper conditions . ThroughWith heuristic approaches come increased risk of error, and given the potential importance of downstream applications such as function prediction, the need becomes clear to properly evaluate the reliability of homology inference tools. This is in itself not a trivial problem, since such benchmarking ideally should involve a ‘gold standard’ where homology status—whether shared common ancestry holds or not—should be known with perfect certainty, which is in principle never the case.The existence of well-conserved ‘building blocks’ of protein sequence and structure, as in domain/gene families where in many cases subtle sequence similarity is supported by clearer similarity of the slower-evolving protein 3D structure , makes fSeveral issues in creating benchmarking datasets have been discussed earlier  describeMore recent developments in homology inference involve profile-based tools for detecting remote homologies, using profile-specific score matrices (PSSMs)  or otherAdditionally, we applied our benchmarking method to all three major domain family databases: SUPERFAMILY  separately. In the case of Pfam, consecutive repeat/motif-type domains, were collapsed to a single instance as in Supplementary Table S2 for details on the number of pairs generated for the final benchmark dataset.For the specific benchmark dataset, all sequences from a specific set of genomes were included, chosen to represent the span of (model organism) diversity while remaining small enough to be manageable (Supplementary Table S3 for details). The recently developed DELTA-BLAST  using each of the profile-based homology search tools CS-BLAST , HHSEARCTA-BLAST , in caseThree challenging homology benchmarks were set up using protein domain architectures based on either the Pfam, SUPERFAMILY, or Gene3D domain definitions. True homologs were defined as multi-domain protein pairs with identical domain architecture, while true non-homologs were randomly picked as multi-domain proteins pairs with no domain in common. The main advantage of using protein domain databases instead of protein structure databases is that also domains with unknown structure are included, such as domains with low sequence complexity. The benchmarks contain 455, 330 and 339 architectures for Pfam, SUPERFAMILY, or Gene3D, respectively. Protein pairs for these architectures were then sampled from 16 species to build a benchmark set of 5245, 5047 and 5656 homologous protein pairs, respectively, with equal numbers of non-homologous protein pairs sampled as well.Supplementary Figure S1A–C. The same overall trends were replicated. Supplementary Figure S2 show corresponding method performance on the benchmark at different specified E-value cutoffs.In total, seven homology search methods were benchmarked: the three profile search tools CS-BLAST, HHSEARCH and PHMMER, as well as the four single sequence search tools FASTA, NCBI-BLAST, UBLAST and USEARCH. To make a fair comparison, we ran all tools with single sequence queries, that is searching the proteins of each benchmark pair against each other in a 1 to 1 setup. Comparing the accuracy  of the tested search tools on all three benchmarks  shows thHow different are the three benchmarks? As they are all mapped to UniProt identifiers, we can compare how often a pair in two benchmarks have the same homology or non-homology status. Restricting to protein pairs present in all three databases controls for difference in coverage, as well as somewhat for differences in hierarchical granularity. Agreement between the databases with respect to homology status reflects the extent to which their differing source data, methodologies and curation efforts uncover the same underlying biological entities, even though it does not guarantee that the domain architectures are identical. As seen in As a complement to benchmarking method accuracy, we also benchmarked run time by applying each tool to 100 randomly chosen protein pairs (repeated 10 times to achieve robust run time estimates), as shown in Given the role of homology inference in genome-scale biology, validation and comparative benchmarking of the tools in use is important, even where it is difficult in both theory and practice to construct such benchmarks so that they will reflect the issues that may come into play in ‘live’ applications. We previously described an extensible strategy for such benchmarking and applied it to the then state-of-the-art of homology inference methods. In the present work, we have updated this approach and applied it to the ‘next generation’ of such methods. We have shown these benchmark results to be robust to the choice of underlying domain definitions, and we make the method available in script distribution for bioinformaticians seeking e.g. to optimize their particular analysis pipelines.From our benchmark we observe that most profile methods have similar accuracy, with top performance from CSBLAST and the HMMER 3 protein search application PHMMER, whereas the speed-optimized FASTA and UBLAST/USEARCH are substantially less accurate. All profile-based methods outperform ‘classic’ single-sequence homology inference tools in terms of accuracy, but some of them do this with great sacrifice of speed.Additionally, we show that the three most widely used protein domain definition schemes are similar with regards to which conclusions on protein full-length homology or non-homology they lead to, implying that the differences between them with regards to source data, curation or methods chiefly lead to differences in coverage and granularity, but not so much to differences in what evolutionary entities end up classified as domain families. Consequently, analysis results from one generally transfers well to the others.It is important to note that development of tools do not take place in a vacuum separated from curation and compilation of protein domain databases. It is therefore conceivable that currently unknown classes of protein folds exist where method performance is different. However, it is likely that most existing folds already are known .As stated previously, this benchmark leaves out recent developments (


In vitro experiments were adopted to measure proliferation and apoptosis in HCC cells after CDK5 mRNA was inhibited in the HCC cell lines HepG2 and HepB3. Based on immunohistochemistry, CDK5 expression levels were notably increased in HCC tissues (n=171) compared with normal , cirrhosis , and adjacent non-cancerous liver  tissues. The up-regulation of CDK5 was associated with higher differentiation (P<0.001), metastasis (P<0.001), advanced clinical TNM stages (P<0.001), portal vein tumor embolus (P=0.003) and vascular invasion (P=0.004). Additionally, TCGA data analysis also revealed significantly increased CDK5 expression in HCC compared with non-cancerous hepatic tissues (P<0.001). The pooled standard mean deviation (SMD) based on 36 included datasets  indicated that CDK5 was up-regulated in HCC . The area under the curve (AUC) of the summary receiver operating characteristic (SROC) curve was 0.88. Furthermore, CDK5 knock-down inhibited proliferation and promoted apoptosis. In conclusion, CDK5 plays an essential role in the initiation and progression of HCC, most likely via accelerating proliferation and suppressing apoptosis in HCC cells by regulating the cell cycle and DNA replication pathways.To investigate the clinical role and biological function of cyclin-dependent kinase 5 (CDK5) in hepatocellular carcinoma (HCC), 412 surgically resected tissue samples  were obtained and analyzed with immunohistochemistry. The diagnostic and prognostic values of CDK5 expression levels in HCC were clarified. Moreover, RNA-seq data or microarray datasets from The Cancer Genome Atlas (TCGA)  or other public databases  regarding CDK5 in HCC were extracted and examined. Several bioinformatic methods were performed to identify CDK5-regulated pathways.  Ranked as the fifth common type of cancer worldwide, hepatocellular carcinoma (HCC) ranks as the third cause of cancer-related deaths . In partin vitro and assessed HCC cell proliferation and apoptosis.As one of the members of the CDK family, CDK5 acts as an important regulator of cell division cycle and was first discovered and reported in 1992 , 11. In P<0.001) (Table P<0.001) for CDK5 protein to diagnose HCC, which indicated a certain value for clinical diagnosis of HCC. HCC patients with metastasis (n=81), portal vein tumor (n=45), vascular invasion (n=52) and advanced TNM stage (n=123) exhibited prominently increased CDK5 expression (P<0.01) , cirrhotic tissues (n=37), adjacent non-HCC liver tissues n=171) to HCC tissues (n=171)  , increased in males (n=250) compared with females (n=121) , increased in pathologic stages III-IV (n=90) compared with pathologic stages I-II (n=257) , and increased in T3-T4 stage (n=93) compared with T1-T2 stage (n=275) . Nevertheless, there are no significant differences between CDK5 expression level and other related pathological subgroups, such as race, relative family cancer history, tumor status, histological grade, N stage, M stage, and vascular tumor cell type  Figure , Table 2) Figure . CDK5 expe Table .P<0.001) by the random-effects model  and adjacent non-tumor tissues n=1995), from online databases . All included datasets are summarized in Table 4 and adj95, from Kaplan-Meier plots were adopted to analyze the survival differences between low and high CDK5 expression levels with the cutoff value defined by the median CDK5 expression level Figure . The ploAfter the calculation described above, 4824 diﬀerently expressed genes (DEGs) were obtained when considering a stringent threshold of |log2FC|>1 and Padj<0.05 Figure . Then, tP=0.001)  Figure . A fluor) Figure . To conf) Figure .in vitro study found that CDK5 could inhibit cell growth and induce apoptosis in HCC cell lines, which might be the mechanism by which CDK5 critically impacted the initiation and development of HCC.The estimated worldwide incidence of liver cancer is 626,000 cases a year. Greater than 50% of cases are from China. Approximately 745,000 people die from HCC yearly worldwide , 21. HCCP<0.001). However, CDK5 expression exhibited no significant differences between cirrhotic and normal tissues in the liver based on immunohistochemistry, indicating that CDK5 is specifically over-expressed in HCC and providing a new marker to distinguish HCC from other hepatic diseases, such as cirrhosis, thus improving the diagnosis accuracy of HCC. Furthermore, from the meta-analysis results of TCGA and other open databases, CDK5 expression in HCC was significantly increased compared with non-HCC liver cancer. Furthermore, ROC analysis was performed in our immunohistochemistry study and revealed that the CDK5 expression level was most useful in the diagnosis of tumor metastasis followed by tissue types, TNM stage, size, embolus and vaso-invasion. These results provide effective target molecules for an accurate diagnosis of HCC compared with other tissue types and to predict HCC progression. Similarly, ROC analysis results of data from the TCGA database confirmed that CDK5 could play an effective role in distinguishing HCC from normal tissues.Our study first demonstrated a significant value of CDK5 in the clinical diagnosis of HCC. CDK5 expression is up-regulated in HCC compared with normal tissues based on immunohistochemistry performed in our study. A similar pattern was revealed by the high-throughput RNA-seq analysis. Therefore, CDK5 over-expression is likely associated with the occurrence of HCC, and further studies should be performed regarding the role of CDK5 expression in HCC diagnosis and individualized treatment. Moreover, compared with cirrhotic and para-carcinoma tissues in the liver, CDK5 expression was increased in HCC  and disease-free survival (DFS). Thus, CDK5 may effectively predict HCC prognosis. However, our RNA-seq results demonstrated that CDK5 could act as a statistically effective HCC prognostic biomarker. Given that the algorithm used in these methods differed, the clinic value of CDK5 in HCC prognosis requires further investigation. In addition to its clinical value, the mechanism by which CDK5 regulates the initiation and development of HCC requires further study.in vitro experiments. In our in vitro experiment, cell proliferation was inhibited in the CDK5-siRNA group, suggesting that CDK5 promotes cell proliferation and subsequently triggers HCC progression. In addition, HCC cell apoptosis increased when CDK5 expression was suppressed, indicating that CDK5 down-regulation induces the low apoptosis rates. Of note, three different methods were adopted to detect the proliferation of both HCC cell lines in our in vitro study, revealing the same trend of cell proliferation. In addition, two different methods were performed to measure apoptosis in both HCC cell lines, revealing a similar trend in cell apoptosis. Thus, the results of our in vitro experiment are reliable. Similarly, Liu JL. Et al.’s study on CDK5 and lung cancer revealed a similar CDK5 proliferation and apoptosis trend in lung cancer cell lines when CDK5 activity was suppressed by siRNA [Based on bioinformatics methods, we hypothesize that CDK5 exercises its functions via several proliferative signaling pathways. We confirmed our hypothesis in a series of by siRNA . A paradby siRNA . As demoby siRNA . CDK5 acby siRNA . Regulatby siRNA . In otheby siRNA . Feldmanby siRNA . In addiby siRNA . Sustainby siRNA . Based oIn summary, CDK5 plays an essential role in HCC initiation and progression, most likely via accelerating proliferation and suppressing apoptosis in HCC cells.http://www.who.int/en/). The age of all the patients ranged from 28 to 76 years . All clinicopathological information was obtained from medical records and summarized in Table In the present study, 412 surgically resected tissue samples were obtained from the First Affiliated Hospital of Guangxi Medical University . The 412 tissues included 33 normal liver tissues, 37 cirrhosis tissues, 171 adjacent non-HCC liver tissues and 171 primary HCC tissues. HCC was diagnosed according to WHO classification of tumors of the digestive system ; 4 implied moderately positive (++) and 5–6 implied strongly positive (+++). Any score greater than 3 in the present study was considered to indicate positive expression in this study.http://cancergenome.nih.gov/). The CDK5 expression data consist of individual 374 HCC samples and 50 normal controls. Clinicopathological parameters, including age, gender, tumor status, race, relative family cancer history, histological grade, TNM stage, pathological T stage, pathological N stage, pathological M stage and vascular tumor cell type, were also estimated. The data above were used to assess the correlation between CDK5 levels and prognosis as presented in the results.CDK5 expression was analyzed by file data downloaded from the TCGA database (https://www.ncbi.nlm.nih.gov/geo/), ArrayExpress (http://www.ebi.ac.uk/arrayexpress/), and Oncomine (https://www.oncomine.org/resource/login.html) databases. The following search words were employed:  AND (hepatocellular OR liver OR hepatic OR HCC).To further examine the CDK5 expression pattern in HCC, we collected HCC-relevant RNA-seq and microarray datasets from GEO (http://www.bioconductor.org/packages/release/bioc/html/limma.html) to identify DEGs between liver HCC and non-tumor tissues. DEGs were selected based on the following criteria: Padj<0.05 and |log2 Fold Change| (|log2FC|) >1. Next, WGCNA, an algorithm for the identification of co-expression gene modules, was performed to compute a set of genes related to CDK5. The process was accomplished using the WGCNA package of the R language (https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/). For CDK5-related genes, GO and KEGG pathway analyses were performed by the online bioinformatic tool The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 and visualized by the R package ‘GOplot’ and ‘ggplot2’.The RNA-Seq data of liver HCC downloaded from TCGA were analyzed using the Limma package of R language . CDK5-siRNA was obtained from Sangon Biotech  .Cell viability was measured by fluorimetric detection of resorufin. The procedure was performed per the manufacturer’s instructions. After transfecting CDK5-siRNA in HepG2 and HepB3 cell lines, cell viability was assayed at 0, 5, and 10 days and compared with mock controls and scrambled siRNA controls.To further verify the cell viability assay data obtained as described above, cell proliferation was measured using a colorimetric tetrazolium (MTS) assay.A synthetic rhodamine-labeled caspase-3/7 substrate  was used to measure caspase-3/7 activity immediately after the detection of the cell viability as described above. The procedure was performed per the instructions of the manufacturer.The impact of CDK5 siRNAs on apoptosis in cell lines was assayed using Hoechst 33342 and propidium iodide (PI) double fluorescent chromatin staining as described in our previous study -19. BrieSPSS 22.0  was applied for statistical analysis of IHC results. Pearson Chi-Square tests was used to evaluate the significance of the role CDK5 in the HCC pathological categories. Pearson Chi-Square tests were also performed to compare CDK5 expression based on the parameters of age, gender, tumor stage (TNM), lymph node metastasis and distal metastasis. The associations between CDK5 expression levels and the clinicopathological characteristics were evaluated using Spearman’s correlation. The diagnostic value of CDK5 was identified by employing ROC. P-values less than 0.05 indicated a statistically significant difference.P<0.05.Regarding data from the TCGA and other public databases, SPSS 22.0 was also used for statistical analysis. R, OriginPro 2017 , and GraphPad Prism 5  were used to plot figures. Data were presented as mean±SD in each of the datasets. The independent-samples T test was used to compare the differential CDK5 expression level in different patients . Similarly, CDK5 expression level in clinicopathological parameters, such as tumor stage (TNM), age, gender, histological stage, and race, were analyzed by independent-samples T test separately. ROC was employed to identify the diagnostic value of CDK5 protein in HCC. Statistical significance was determined at To obtain a comprehensive perspective on CDK5 expression, we integrated multiple source data in the form of meta-analysis using STATA 12.0 . The total SMD was computed. When SMD>0 and its 95% CI did not cross, an integer of 0 indicated that CDK5 in tumors is significantly overexpressed compared with adjacent non-tumor tissues. To further study the comprehensive efficiency of CDK5 in distinguishing tumor from non-tumor tissues, we generated SROC curves and calculated the AUC value with 95% CI, sensitivity and specificity.In vitro experimental data were analyzed by SPSS and graphed using GraphPad Prism 5  directly. Appropriate graphs  were generated to represent the relationship between CDK5 and proliferation as well as HCC cell apoptosis.


Three domain-swapped cytochrome cb562 dimers formed a unique cage structure with a Zn–SO4 cluster inside the cavity. c-type cytochromes and myoglobin form oligomers by domain swapping. Herein, we show that a four-helix bundle protein cyt cb562, with the cyt b562 heme attached to the protein moiety by two Cys residues insertion, forms a domain-swapped dimer. Dimeric cyt cb562 did not dissociate to monomers at 4 °C, whereas dimeric cyt b562 dissociated under the same conditions, showing that heme attachment to the protein moiety stabilizes the domain-swapped structure. According to X-ray crystallographic analysis of dimeric cyt cb562, the two helices in the N-terminal region of one protomer interacted with the other two helices in the C-terminal region of the other protomer, where Lys51–Asp54 served as a hinge loop. The heme coordination structure of the dimer was similar to that of the monomer. In the crystal, three domain-swapped cyt cb562 dimers formed a unique cage structure with a Zn–SO4 cluster inside the cavity. The Zn–SO4 cluster consisted of fifteen Zn2+ and seven SO42– ions, whereas six additional Zn2+ ions were detected inside the cavity. The cage structure was stabilized by coordination of the amino acid side chains of the dimers to the Zn2+ ions and connection of two four-helix bundle units through the conformation-adjustable hinge loop. These results show that domain swapping can be applied in the construction of unique protein nanostructures.Protein nanostructures have been gaining in interest, along with developments in new methods for construction of novel nanostructures. We have previously shown that  Domain swapping has attracted attention as a mechanism of protein oligomerization, and reports on domain swapping have been increasing.9 In domain swapping, a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule.10 We have previously shown that small spherical heme proteins, c-type cytochrome (cyt) proteins and horse myoglobin (Mb), form oligomers by domain swapping.11 In the dimer and trimer of horse cyt c, the C-terminal α-helix domain swapped with the corresponding region of other molecules. In dimeric Pseudomonas aeruginosa (PA) cyt c551 and dimeric Hydrogenobacter thermophilus (HT) cyt c552, the region containing the N-terminal α-helix and heme was swapped.d Previously, we proposed that the unstable loop region has a tendency to become a hinge loop for domain swapping in cyt c family proteins.11 In domain-swapped dimeric horse Mb, each active site consisted of two different protomers, and new long α-helices were formed by the E and F helices and the EF-loop of the original monomer.11 Although apoMb formed a dimer, a large amount of apoMb dissociated to monomers by incubation at 37 °C for 30 min, whereas holoMb oligomers did not dissociate under the same conditions.11 These results indicated that the Mb dimer was stabilized by heme binding to the protein.Protein nanostructures for development of artificial biomaterials have been constructed under various methods, such as chemical modification,b562 from E. coli is a relatively small (MW: ∼12 000) heme protein responsible for electron transfer in the periplasm.12 Owing to its simple four-helix bundle structure, cyt b562 is frequently used as a model for studying protein stability and folding.13 Cyt b562 from E. coli is composed of 106 amino acids, and four α-helices are arranged in an antiparallel orientation.14 Met7 and His102 are coordinated to the heme iron in cyt b562, where the heme may dissociate from the protein moiety. A c-type cyt b562 (cyt cb562) has been constructed by Barker and co-workers by replacing Arg98 and Tyr101 with Cys residues and introducing two covalent thioether bonds between the heme and protein moiety.15 Gray and co-workers have shown that the covalent attachment of the heme to the protein moiety in cyt cb562 increases its protein stability without perturbation in the structure and folding process from those of the wild-type protein (cyt b562).16Cyt cb562 nanostructures by introducing intermolecular interaction sites at the protein surface.17 A cyt cb562 variant with two metal-chelating bis-His motifs at its surface has been shown to assemble into a tetramer, trimer and dimer through Zn2+, Ni2+ and Cu2+ coordination, respectively.17 Cage-like tetrahedral dodecamers have been constructed in the crystal lattice of another cyt cb562 variant.17 A cyt cb562 derivative with two additional Cys residues at its surface has been shown to assemble into a tetramer with a cryptand-like topology through selective formation of four interfacial disulfide bonds.17 A computationally designed dimer of cyt cb562 has been arranged by Zn2+ coordination into 1D helical nanotubes, and 2D or 3D crystalline arrays.6Tezcan and co-workers have constructed cyt 18 demonstrating that domain swapping may be utilized to design artificial heme proteins. In this study, we obtained domain-swapped cyt cb562 oligomers, and found that the two helices (helices 1 and 2) in the N-terminal region of one protomer and the other two helices (helices 3 and 4) in the C-terminal region of the other protomer interact in the dimer. In the crystal, three cyt cb562 dimers formed a unique cage structure with a Zn–SO4 cluster inside the cavity, showing that domain-swapping may be utilized to construct unique nanostructures.Recently, we have constructed a heterodimeric Mb with two different active sites by modifying the interface of two protomers in the domain-swapped wild-type dimer,E. coli cyt b562 solution changed its colour from red to black by an addition of acetic acid up to 40% (v/v) (pH 1.9) to its neutral pH solution, although no precipitation was observed. Cyt b562 has been reported to denature at pH 2.3.19 Therefore, the colour change was ascribed to the heme dissociation from the cyt b562 protein moiety in the presence of 40% (v/v) acetic acid, causing change in the heme coordination. After lyophilization of the cyt b562 solution, the protein powder was dissolved in 50 mM potassium phosphate buffer, pH 7.0, and analysed by size exclusion chromatography. A small peak was observed at ∼11.1 mL in the elution curve, in addition to the monomer peak at ∼12.7 mL  and subsequent dissolution of the obtained precipitate with buffer  of dimeric cyt cb562 ,20 and to that of cyt b562 (205 mV)  was similar to that of the cyt cb562 monomer. Dimeric cyt cb562 exhibited a similar high redox potential as the monomer, suggesting that the active site structure of the dimer is similar to that of the monomer.Cyclic voltammetry (CV) was performed to determine the midpoint redox potential (t cb562 . The Em(205 mV) . The Emcb562 were performed to investigate the thermodynamic properties of the dimer. A positive peak was observed at 83.9 °C in the DSC thermogram of the cyt cb562 monomer .13 However, an additional small negative peak was observed at 50.1 °C in the DSC thermogram of the cyt cb562 dimer  during the dissociation. The ΔH value for the dissociation of the cyt cb562 dimer to monomers was obtained as –13 ± 2 kcal mol–1 (per dimer), showing that the dimer is enthalpically disfavoured compared to the monomer.Differential scanning calorimetry (DSC) measurements of oxidized monomeric and dimeric cyt  monomer . We assi62 dimer . No peakC Fig. S3. Therefocb562. The 1.85 Å resolution structure of dimeric cyt cb562 exhibited a domain-swapped structure   and dimer. Residues in the N-terminal region (Ala1–Asp50) before a protomer hinge loop (Lys51–Asp54) and residues in the C-terminal region (Ser55–Arg106) after the hinge loop in the other protomer in the dimer were compared with the corresponding structural region of the monomer. The rmsd values were less than 0.36 Å, indicating that the corresponding structures of the monomer and dimer were similar. The heme orientation and the amino acid side chain positions at the active site of the dimer also overlapped well with those of the monomer  . The two Fig. S4A. We calc Fig. S4B. Met7 an Fig. S4B, althoug Fig. S4B.cb562 formed a hollow cage-like structure in the crystal (D3 symmetry with one 3-fold and three pseudo 2-fold axes. The outer diameter of the cage was 55–60 Å (2+ and seven sulfate (SO42–) ions was encapsulated in the cage   were also observed inside the cavity  measurements of oxidized dimeric cyt cb562 were performed with an addition of ZnSO4, ZnCl2 and Na2SO4 at pH 5.5, at which the crystal was obtained (cb562 with an addition of ZnSO4 (Zn2+ and SO42– ions) (4.6 ± 0.3 nm) compared to that with an addition of ZnCl2 (Zn2+ and Cl– ions) (3.4 ± 0.4 nm) or Na2SO4 (Na+ and SO42– ions) (3.1 ± 0.6 nm), indicating that the dimers interacted among each other in solution in the presence of Zn2+ and SO42– ions, although a defined cage structure was not constructed.To investigate the interaction of dimeric cyt obtained . The meacb562 solution and cross-linked cyt cb562 to investigate the intermolecular interaction of cyt cb562 in solution in more detail. After the reaction of the cyt cb562 dimers with BS3 in the presence of Zn2+ and SO42– ions, the solution was analysed by SDS-PAGE  to the oxidized dimeric cyt SDS-PAGE . Four baectively . These r11 In c-type cyts and Mb, the active sites of the domain-swapped oligomers are constructed with two protomers.11 The heme is coordinated with the amino acids from different protomers in dimeric c-type cyts,e which may stabilize the domain-swapped structure. In cyt cb562, less domain-swapped dimers dissociated to monomers at 4 °C compared to cyt b562  is –42 kJ mol–1, whereas that of oxidized cyt b562 is –30 kJ mol–1.16 The folding free energy change of oxidized HT cyt c552 exhibits a greater negative value than that of oxidized horse cyt c ),22 whereas the dissociation temperature of the domain-swapped HT cyt c552 dimer (92 °C)11 is higher than that of the horse cyt c dimer (58 °C).11 Since a domain-swapped dimer shares a similar three-dimensional structure with its monomer, excluding the hinge loop, the folding free energy change of the dimer may correlate with that of the monomer. In fact, dissociation of a domain-swapped dimer to monomers has been suggested to occur via significant unfolding of the polypeptide for many proteins, including RNase A, cyanovirin-N, Stefin A, MPro-C and p13suc1.23 Taking these results into consideration, stabilization of the domain-swapped dimer on dissociation to monomers may correspond to the negative value in the folding free energy change of its monomeric protein.Domain swapping has been observed in various heme proteins.2 Fig. S2. It has H value for the dissociation of the cyt cb562 dimer to monomers was obtained as –13 kcal mol–1 (per dimer) ,11 whereas that of the PA cyt c551 dimer, HT cyt c552 dimer and Aquifex aeolicus cyt c555 dimer are ∼0, +14 and –14 kcal mol–1 (per dimer), respectively.e Although many other factors including solvation may affect the ΔH value, the number of hydrogen bonds at the hinge loop decreased in cyt cb562 by the dimerization   . The ΔH ) Fig. S7.24 This cb562 ), and presumably refolded by dissolution of the precipitate with buffer  of a protomer and the other two helices in the C-terminal region (helices 3 and 4) of the other protomer interacted between each other in domain-swapped dimeric cyt cb562 . Althougcb562  and S4B†ed dimer . We havemonomers . Howevercb562 was formed by three dimers in the crystal  and Glu (Glu4 and Glu8), together with the amino and carbonyl groups of Ala1, were coordinated to Zn2+ ions in the cavity of the domain-swapped cyt cb562 cage . The cavity size of the domain-swapped cyt cb562 cage was calculated as 1860 Å3 by the program, VOIDOO,33 using a probe radius of 1.4 Å and excluding the Zn2+ and SO42– ions in the model; whereas, it was 32 740 Å3 for the cage structure of the cyt cb562 surface mutant. The interaction of the Zn2+ ions through the SO42– ions may be a factor in the small cavity size of the domain-swapped cyt cb562 cage.Although protein cages are potentially useful for many applications, such as drug and gene carriers,2+ ion binding sites have been detected in the cyt cb562 surface mutant cage.17 The Zn2+ ion coordinating non-modified residues  in the surface mutant cage17 also coordinated to the Zn2+ ions in the domain-swapped dimer cage. Especially, the Zn7 site with Ala1, Asp12 and Asp39 of the domain-swapped dimer cage corresponded well to the Zn2+ ion binding site with Ala1 and Asp39 of the surface mutant cage  in dimeric cyt cb562, the relative positions of the two units could be adjusted, whereas the entropy loss on cage formation by three domain-swapped dimers may be smaller than that by six monomers. These properties may have guided the dimers to form a cage structure.Monomeric cyt  Fig. S4A. Therefob562 and cyt cb562 by treatment with acetic acid. Dimeric cyt cb562 was more stable than dimeric cyt b562 at 4 °C, showing that the dimer stability increases by the heme attachment to the protein moiety. According to X-ray analysis, dimeric cyt cb562 exhibited a domain-swapped structure, where the two helices in the N-terminal region (helices 1 and 2) in a protomer and the other two helices in the C-terminal region (helices 3 and 4) of the other protomer interacted between each other. These results were in agreement with the previously mentioned proposal that helices 2 and 3 form at the initial cyt cb562 folding stage. Three dimeric cyt cb562 formed a cage structure containing a Zn–SO4 cluster inside the cavity in the crystal. Although no hydrogen bond or hydrophobic interaction existed at the interfaces of the dimers, coordination of the amino acids of cyt cb562 to Zn2+ ions directed formation of the cage structure. The cavity size of the domain-swapped cyt cb562 cage was smaller than that of the cyt cb562 surface mutant cage, where the interaction of the Zn2+ ions through the SO42– ions was important for maintaining the small domain-swapped cyt cb562 cage. The connection of two four-helix bundle units through the hinge loop in the domain-swapped dimer apparently stabilized the cage structure. These results show that domain swapping may be useful for designing artificial proteins with unique nanostructures, by connecting structural units with a conformation-adjustable loop.Oligomers were obtained for cyt