Atmospheric pressure cold plasma has the potential to modify biological chemistry and modulate physical surface properties. Wheat flour was treated by low levels of cold plasma  for 60 or 120 s. There was no change in the total aerobic bacterial count or total mould count as a result of treatment. Treatment did not impact the concentration of total non-starch lipids, or non-polar and glycolipids. However, treatment did reduce total free fatty acids and phospholipids and was dose dependent. Oxidation markers  increased with treatment time and voltage, which confirmed the acceleration of lipid oxidation. Total proteins were not significantly influenced by treatment although there was a trend towards higher molecular weight fractions which indicated protein oxidation and treated flour did produce a stronger dough. This study confirms the potential of cold plasma as a tool to modify flour functionality. All stabilization methods modified the nutritional characteristics of whole wheat bread, especially UV-C and infrared treatments, which had positive effects on proteins and antioxidant activity. Autoclave and microwave treatments increased the total extractable mineral and HCl extractable minerals and decreased extractable phytic acid content. A wide range of different wheat flour treatments  have previously been trialled to stabilise or modify the functionality of wheat flour . Previou3, KBrO3 and ascorbic acid) or oxidising enzymes (e.g. lipoxygenase) have been shown to modify wheat flour  including singlet oxygen, ozone and excited molecular nitrogen . This waCold plasma has the potential to change the chemical composition of wheat flour through radical induced and ozone propagated oxidation and thereby change the functionality of the flour. However, the extent of the changes in the flour needs to be determined and controlled to enhance flour functionality. Therefore, this study was conducted to identify the effect of low doses of cold plasma treatment on wheat flour microbial load, chemical composition (protein and lipid) and dough rheology. A low treatment level was selected to minimise any secondary effects such as the formation of oxidation products that might be aroma active.22.1Triticum aestivum L.) was prepared at Campden BRI , and was matured for 2 weeks. The flour was then stored at −20 °C. All reagents were sourced from Sigma–Aldrich  and were of analytical grade.Bread type wheat flour (2.22 and 0.43 W/cm2). The gas was the surrounding air on the day of treatment.Wheat flour samples (12 g) in petri dishes were treated for 60 s and 120 s using a continuous cold plasma prototype (the system was manufactured through collaboration with the University of Liverpool and Campden BRI and was funded by the EPSRC Impact Acceleration Account Scheme). A plasma discharge was generated using an operating frequency of 9 kHz with an applied power of 40 ± 1 W and 90 ± 1 W for DC power supply input voltages of 15 and 20 V respectively  was diluted tenfold using Maximum Recovery Diluent , and appropriate dilutions were spread plated in triplicate (0.1 mL of dilution) on Plate Count Agar . Plates were incubated at 30 °C for 24–48 h (mesophiles) and 42 °C for 24–72 h (thermophiles).For the detection of moulds, wheat flour (3 g) was diluted ten-fold using MRD  and appropriate dilutions were spread plated in triplicate  on Rose-Bengal Chloramphenicol agar . Plates were incubated aerobically at 25 °C for 3–5 days (limit of detection = 3 cfu/g). After incubation, colonies were enumerated. Results were expressed as log cfu/g of wheat flour.2.4The Bligh and Dyer lipid extraction protocol  was usedThe most abundant fatty acids were palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2) and linolenic acid (C18:3), although others were identified (<5%). The total non-starch lipid was calculated and reported as the sum of the five major fatty acids (% w/w), to maximise comparability and minimise analytical variation that may be introduced by including low concentration lipid components. All analysis was carried out in triplicate.2.4.1The isolated lipid fraction was then fractionated by a SPE process (SPE 1) into neutral lipids, glycolipids and phospholipids. Silica SPE columns  were conditioned using 5 mL hexane followed by 5 mL chloroform. The extracted lipid in chloroform (1 mL) was then added to the top of the silica SPE column and the drained solvent was collected and tested to ensure all lipids were loaded on the column. Chloroform:acetone  (10 mL) was added after the complete elution of the solvent; Neutral lipids were eluted at this step. Acetone:methanol  mixture solvent (15 mL) was added to elute glycolipids. Methanol (10 mL) was added to elute phospholipids. The flow rate of the SPE holder was adjusted to 0.7 mL/min by applying a vacuum manifold . Prepare2.4.2The neutral lipid fraction obtained from SPE 1 was then fractionated by a second SPE process (SPE 2) into non-polar lipids and free fatty acids. SPE columns  were pre-conditioned using 4 mL hexane. The neutral lipids prepared by SPE 1 in chloroform (1 mL) were loaded to the top of the silica SPE column. n-hexane:diethylether  (4 mL) was added, neutral lipids were eluted at this step . Free fatty acids (FFAs) were eluted by a mixture of n-hexane:diethylether  (4 mL). After the complete elution of solvent containing FFAs, methanol (4 mL) was added to wash out remaining polar lipids. After separating free fatty acids from the neutral lipids, the other two fractions obtained from SPE 2 were considered as non-polar lipids.2.4.3m/z, scan rate 500 amu/s and scan time of 0.69 s, source temperature 200 °C). Compound identification was achieved by matching with database MS spectra , and comparing retention time and MS spectra with those of authentic FAME standards . Quantification was achieved using the ratio of each target compound’s peak area relative to that of the internal standard.Crude and fractionated lipid extracts were evaporated to dryness under a nitrogen stream, dissolved in chloroform (1 mL) and stored at −80 °C until analyses. Lipid samples were thawed, combined with an internal standard (40 μg heptadecanoic acid in 20 μl chloroform) and derivatized to fatty acid methyl esters (FAME) with 200 μl of 0.25 M TMSH in methanol (10 min at ambient temperature) . FAME sa2.4.4Wheat flour (0.5 g) was extracted using the Bligh and Dyer lipid extraction methodology with 3 replicate extraction steps. The extracted lipids were dried under nitrogen and dissolved in 1 mL chloroform. The extracted lipid in 1 mL chloroform (200 μl) was then analysed for hydroperoxide value (PV) using the method of 2.5The abundance of n-hexanal was calculated in the sample headspace using solid phase micro-extraction gas chromatography mass spectroscopy (SPME-GCMS) . Headspa2.6g) for 5 min. An aliquot of the supernatant was sealed in a HPLC vial ready for analysis. SE-HPLC analysis was conducted using a Jasco system  operating with a TSK gel G 4000SW column (30 cm × 7.5 mm) and a TSK gel SK guard column (7.5 cm × 7.5 mm). The flow rate of the solvent, 0.1 M phosphate buffer containing 1.1% SDS, was 0.7 mL/min, and detection was performed at 214 nm.Size exclusion high-performance liquid chromatography (SE-HPLC) was used to determine the protein polymer size distribution of the white flour samples. The analysis was performed according to the Profilblé method developed jointly by ARVALIS and l’Institut National de Recherche Agronomique . Flour   was used to analyse the apparent viscosity of flour samples as a function of time, temperature and stirring. Flour (3 g) mixed with water (25 g) based on 12% moisture content  and stirred at a constant speed (160 rpm) during the experiment. Temperature profiles used were: hold for 1 min at 50 °C, heat at 10 °C/min from 50 to 90 °C, hold for 2.5 min at 95oC, cool at 10 °C/min to 50 °C and hold for 1.5 min at 50 °C, pasting curves were performed in duplicate. Only the most extreme treatments were tested by RVA, Reomixer and by SE HPLC.2.7.2A reomixer was selected for analysis of dough rheology as it was able to operate effectively with small sample sizes. The reomixer (Bohlin) consists of an instrumented 10 g planetary pin mixer, a microprocessor unit for signal conditions and software for data acquisition. The reomixer recorded the torque–time trace from the developing dough as described in the manual of methods for wheat and flour testing . Previou2.8post hoc test using IBM SPSS Statistics, version 19 . Probability values lower than 0.05 were considered significant.All experiments were conducted with a fully balanced experimental design, with randomised analysis order and three sample replicates. Comparison between extraction methods was carried out by one-way analysis of variance and Tukey’s HSD 33.1It has been shown in previous studies that cold plasma treatment may reduce microbial viability  and for 3.2The impact of lipid oxidation on bread quality is poorly understood, but many authors  have relWhen comparing the total extractable lipids of cold plasma treated wheat flour with untreated control flour no significant difference was found (data not shown). When the total extractable lipids were fractionated into different lipid classes , no significant difference was found in the non-polar and glycolipids fractions . HoweverThe impact of cold plasma treatment on free fatty acids was due to a reduction in all major fatty acids ; this waIn order to determine the progression of lipid rancidity, primary  and secondary markers of lipid oxidation  were determined. A similar trend was observed for both PV and n-hexanal abundance, in both cases there was a significant increase in the oxidation markers for all of the cold plasma treated wheat flour samples . The dev3.3In addition to the lipids being affected by the oxidative environment, the proteins could also be expected to be altered. Oxidation of wheat flour proteins is believed to directly impact a range of functional properties . The oxiSE-HPLC was used to separate the total grain proteins (monomers and polymers) based on their size distribution, without reduction of disulphide bonds, with five peaks being recognised. The first peak to elute from the column is referred to as F1 and consists of HMW polymers enriched in HMW glutenin subunits (GS), while the F2 peak comprises LMW polymers and is enriched with LMW GS. The F3 and F4 peaks are comprised principally of ω-gliadins and α-, β-, and γ-gliadins, respectively, and the F5 peak comprises LMW proteins including albumins and globulins. The total area under the trace is a measure of the total protein content of the flour. No significant change was found in the total protein levels, but the flour subjected to the highest treatment (20 V for 120 s) had significant changes in the distribution of protein fractions and had a greater F1 fraction and lower F3, F4 and F5 fractions  A. Overal3.4The present study showed that the proportion of F1 + F2/F3 + F4 significantly increased in cold plasma treated flour and that there was enhanced oxidation of the lipids. To investigate if these changes had an impact on the functionality of the flour a series of functional tests were applied to the treated and non-treated flours.To test the general pasting characteristics the samples were assessed using an RVA. The wheat flour sample at the highest level of treatment (20 V for 120 s) showed no significant change in its pasting properties when compared to the untreated control flour . The pas42 and 0.43 W/cm2 for different time periods and the samples assessed for chemical and physical changes. Although cold plasma has been used previously to reduce the viability of microorganisms found in wheat samples, the low treatment levels used showed no reduction in the levels of aerobic mesophiles, thermophiles and associated spoilage fungi, this was presumably due to the low moisture levels and low treatment levels.In conclusion, a bread making quality wheat was milled and then successfully subjected to low level cold plasma treatment using applied powers of 0.19 W/cmThe treatment was effective in creating changes in the lipid components of the flour. Cold plasma treatment was found to result in a voltage and treatment time dependent reduction in non-starch FFAs and phospholipids and progression of oxidation was evidenced by a significant increase in PV and n-hexanal concentration at all levels of treatment, suggesting that cold plasma is resulting in an acceleration of flour oxidation.A shift towards higher molecular weights in the protein profile, as analysed by SE-HPLC, was seen at the higher energy inputs which could be expected to increase the strength of dough made from the flour. This was indeed the case as small scale dough rheology tests (Reomixer) showed that treated wheat flour resulted in stronger dough.Having shown that cold plasma could be effective in enhancing flour functionality it is recommended that consideration is given to optimise the cold plasma treatment and evaluate the process on a wide range of wheat flours, as this was not included in our study, this is especially true of whole wheat flours where acceleration of oxidation may be detrimental due to the higher lipid levels present.Ultimately, this level of control of low levels of oxidation could be critical for enhancing functionality of wheat flour by promoting appropriate dough structure formation for new product development scientists and new ingredient designers.


Background: Single α-helix (SAH) domains bridge two functional domains in proteins. Their force response is poorly understood.Results: Modeling and experiments show that SAH domains unfold non-cooperatively at low forces and maintain an approximately constant force as they unfold.Conclusion: SAH domains act as constant force springs.Significance: SAH domains are important mechanical elements in proteins. Single α-helix (SAH) domains are rich in charged residues  and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (<30 pN) without a characteristic unfolding peak. Molecular dynamics simulations showed that the SAH domain unfolds progressively as the length is increased and refolds progressively as the length is reduced. This enables the SAH domain to act as a constant force spring in the mechanically dynamic environment of the cell. SAH domains appear to be fairly rigid and resistant to bending as demonstrated by a combination of optical trap, small angle x-ray scattering measurements, and modeling . This su1–The biological function of SAH domains is likely to be determined by how rigid and inextensible they are over their length. Therefore, it is important to determine how well the SAH domains maintain their highly helical structure over a long length of sequence. For example, do they act as a stiff spacer or as a weak extensible element that unfolds at low forces before other regions of the protein unfold? To answer these questions, we characterized a predicted 97-residue-long SAH domain from myosin-10 using a combination of approaches. We determined the secondary structure of the peptide and its thermal melting characteristics by circular dichroism and confirmed that it was monomeric by mass spectrometry. We used single molecule atomic force spectroscopy to determine its unfolding properties, and we used molecular simulation to understand the physical properties that underlie its unique structural features. These results provide novel insight into the properties of the SAH domain and suggest new ideas about its biological role.A280 concentration measurements.PCR was used to amplify the sequence for the SAH domain used in these experiments using bovine myosin-10 cDNA as a template . The SAH domain was cloned into the pET28a SUMO  vector to introduce an N-terminal His tag and small ubiquitin-like modifier protein for increased expression and solubility. A tryptophan residue was added to the C terminus to enable 5SAH1 construct, the SAH domain was cloned between the second and third I27 domains. To generate the I275SAH2 construct, an additional SAH domain was cloned into I275SAH1 between the first and second I27 domains.A modified pET3 vector encoding a pentamer of titin I27 domains (His tag-I27-I27-I27-I27-I27-Cys-Cys) was a kind gift from Dr. David Brockwell, University of Leeds . To geneEscherichia coli BL21 Rosetta 2 (Novagen) and purified using nickel-nitrilotriacetic acid affinity and ion-exchange chromatography. For SUMO constructs, SUMO was removed using ULP1 protease before ion-exchange chromatography. The purest fractions were combined and concentrated, resulting in 0.4–1.2 mg/ml protein solutions. Purified protein was dialyzed against 100 mm NaCl, 10 mm sodium phosphate, pH 7.4 and snap frozen in liquid nitrogen for long term storage at −80 °C.All proteins were expressed in m) were dialyzed  overnight against 50 mm ammonium acetate, pH 7.4 and analyzed by TOF MS analysis (The University of Leeds Mass Spectrometry Facility).Protein samples  constructs or 100 mm NaCl, 10 mm sodium phosphate, pH 7.4 for SAH domain). Data were collected every 1 nm with 30-s averaging time with each measurement being an average of two repeated scans. Data presented are averaged from at least three separate measurements of different protein preparations. Thermal measurements were performed in a temperature range from 10 to 85 °C with 0.7 °C/min heating rate with data acquisition every 1 °C and a 20-s averaging time. The sample cooling rate prior to measurement of refolded protein was ∼ 2 °C/min.CD measurements were performed on an Applied Photophysics Chirascan CD spectropolarimeter with a 0.1-cm-path length quartz cuvette in CD buffer  of the “empty” I275 construct was subtracted from those of I275SAH1 and I275SAH2 constructs. The mean residue molar ellipticity of proteins was calculated as described (nπ* transition at 222 nm ([θ222]) as described previously  were incubated on freshly cleaved template-stripped gold for ∼10 min. Experiments were performed either directly in this solution or after replacement with Tris buffer.Protein constructs at ∼0.1 mg/ml in Tris buffer (150 mkc) were typically 35–45 pN/nm. Force-extension traces were collected at a velocity of 1000 nm/s over a distance of 500–1000 nm with a sampling rate of 5 kHz and a maximum loading force of ∼1.5–3 nN. The position of the tip on the substrate was manually changed every few hundred traces.All AFM work was carried out on an Asylum Research MFP-3D instrument operated at room temperature. Cantilevers  were calibrated prior to collection of force-extension traces using the thermal noise method ; spring single protein molecule, which had been picked up by its N terminus, only traces where five I27 domains were seen to unfold followed by a detachment peak were used. Traces with multiple detachment peaks or where the contour length up to the detachment peak was unphysical  were excluded. Furthermore, only those traces showing a reasonable fit to a wormlike chain (WLC) model were used. The WLC model links force (F) to extension (x) and is commonly used to describe the elasticity of single polypeptide chains  and cl, the contour length (the length the polymer approaches at high force). Using an initial global fit to the five I27 unfolding events, only those traces showing an increase of contour length (cl) on unfolding titin I27 domains of 28 ± 1 nm and a persistence length (p) of 0.4 ± 0.1 nm (uF)) and the peak-to-peak distance (P2P) were recorded. WLC parameters for a fit up to 100 pN on the first I27 unfolding peak were also recorded. The fit was limited to 100 pN to avoid the onset of the “hump” feature due to I27 unfolding intermediates  were calculated from the expected length of unfolded SAH domains and any linker sequences between I27 and SAH domains present in each construct, using 0.38 nm/residue for an unfolded peptide residue    showed t97, and Gly97 or from the Protein Data Bank structure in the case of I27 followed by a short (100-ps) dynamics run. Perfectly α-helical conformers were created by setting internal dihedral angles to ϕ = −47° and φ = −57°. Zero force simulations lasted at least 100 ns.Starting structures for zero force simulations were prepared by performing a steepest descent minimization (1000 steps) from artificially generated perfectly α-helical conformers of myosin-10 SAH domain, Ala−6 nm/ns) due to computational time constraints, and this results in higher values for the unfolding forces in silico compared with experiments.Constant velocity force spectroscopy experiments were mimicked in MD simulations by applying an external force between the N-terminal nitrogen atom and C-terminal carbonyl carbon atom as the “cantilever” and moving this away at constant velocity. The value for the single molecule force constant for the springs (30 pN/nm) is realistic and comparable with that of the cantilever used in single molecule force spectroscopy experiments. A range of velocities was used (from 10 to 0.01 nm/ns). These are several orders of magnitude higher than those used in the real experiment ) of at least 8 nm had been reached. Wordom (version 21) was used to analyze the simulation trajectories . This sequence  was originally predicted to form coiled coil. However, we showed that a peptide containing the first 36 residues (808–843) is not coiled coil but behaves as a SAH domain (B) shows that although there are fewer charged interactions downstream of the first 36 residues charge-charge interactions are still common, suggesting that this entire region may act as a SAH domain. The distal part of this sequence (residues 883–934) was recently shown to form an antiparallel coiled coil in an isolated peptide, but residues downstream of residue 909 were required for its formation  and the tail domain A. This sH domain . The firH domain . In thatH domain B shows tormation . Therefoin vitro. The CD spectrum at 10 °C showed the characteristic strong double minima at 208 and 222 nm typical of an α-helix (C). The peptide was estimated to be ∼67% helical from the value obtained at 222 nm; this is similar to the helical content measured for the first 36 residues of this SAH domain in earlier experiments (D), and this effect was reversed on cooling to 10 °C at which the helical content returned to at least 80% of its original value before thermal denaturation (C), consistent with the behavior of SAH domains  was dimeric with a mass of 25.7 kDa (compared with the predicted molecular mass of 12.9 kDa for the monomer).Mass spectrometry and UV CD experiments showed that as predicted the 97-residue myosin-10 peptide did behave as a SAH domain  α-helix C. The peeriments D, and thturation C, consis5SAH1 and I275SAH2, respectively; Fig. 2A).To perform single molecule force spectroscopy experiments, the SAH domain was inserted between titin I27 domains. The I27 domain was used as a reference protein in these experiments as its unfolding profile in single molecule force spectroscopy experiments has been well characterized . Two con5SAH1 and I275SAH2 constructs. Their CD spectra are complex as there are contributions from both I27 domains, which have a β-sandwich structure (5 construct (B) decreases the mean residue ellipticity value at 222 nm as expected for the insertion of an α-helix into the I27 construct. Thus, both I275SAH1 and I275SAH2 contain a mixture of β-sheet and α-helix as expected if the SAH domain remains helical. Subtraction of the I275 spectrum from those for I275SAH1 and I275SAH2 revealed typical α-helical spectra with two minima present at 222 and 208 nm and a maximum at 195 nm (C). The magnitude of these minima is indistinguishable from that observed for the isolated SAH domain from myosin-10 (D).Before carrying out the single molecule force spectroscopy experiments, we first used CD to confirm that the SAH domain remained helical when inserted between I27 domains in the I27tructure , and SAHonstruct B decreast 195 nm C. The mayosin-10 D.5SAH2 was able to fully refold after thermal melting (E), whereas I275 was not (F). In a mixture of monomeric I27 domains and SAH domains in a ratio of 5:2 (the same ratio as in the I275SAH2 construct), both peptides were able to refold after thermal melting as the CD traces superpose (G). In contrast, a mixture of I275 and SAH domains in a ratio of 1:2 (as in the I275SAH2 construct) showed that I275 does not refold after melting. Note that melting of I275 decreases the MRE222nm value (becomes more negative), whereas melting of the SAH domain increases it. Hence, in this case, the folded (10 °C) and unfolded (85 °C) traces superpose. On refolding, the 222 nm value decreases due to the refolding (decrease in MRE222nm) of the SAH domain only. The inability of I275 to refold likely results from subdomain swapping between the concatenated I27 domains that interferes with the refolding of each individual domain . This approach ensures that only single molecules, which have been picked up by their ends, are included in our analysis (5), one (I275SAH1), or two (I275SAH2) identical SAH domains allows us to be sure that we have correctly identified the contribution of the SAH domain to the trace.As the I27analysis . Importacl from a WLC fit up to the first I27 unfolding event in I275 was 47 nm . The average contour length from a WLC fit to the first I27 unfolding event increased from 47 (I275) to 77 nm (E). This increase in contour length is in line with the calculated increase in length that would result from addition of an unfolded SAH domain (G–I) increased the distance to the first I27 unfolding peak even further (G–I), increasing the calculated contour length to 120 nm (F). These data show that the SAH domains unfold before the first I27 domain unfolds. The lack of an additional unfolding peak is strongly suggestive that the SAH domain unfolds non-cooperatively.Importantly, introducing one SAH domain into the I27ing peak , D–F. Thto 77 nm E. This iH domain . SimilarH domain , G–I inc further , G–I, ino 120 nm F. These 5 construct, again supporting our conclusion that the SAH domain unfolds before the first I27 domain. The measured P2P , uF  for all three constructs were all in good agreement with previous studies  as used in these experiments. In addition, we used the behavior of polyalanine (Ala97) as a model of a “normal” α-helix and polyglycine (Gly97) as a model random coil. The SAH domain and the Ala97 and Gly97 peptides were all given α-helical structures at the start of each simulation.To confirm our experimental findings that the SAH domain unfolds non-cooperatively at forces below that at which the I27 domains unfold, we used MD simulations. We compared the dynamic behavior of the myosin-10 SAH domain with that of the monomeric I27 domain . In single molecule force-extension simulations, a saw-toothed curve was observed , consistent with experimental observations. A small change in length as force increased was followed by a large change in length associated with a collapse in force as the protein unfolds .The MD simulations showed that the titin I27 domain maintained a structure close to its initial crystal structure throughout the lifetime of the simulation in the absence of any applied force or length constraint as expected  with an average helicity of 74 ± 6% in good agreement with our experimental finding. The stability of the SAH domain is evidenced by the limited change in end-to-end distance (rNC) as a function of time. In single molecule force-extension simulations, the initial rise in force was lower than that observed for I27 and was not followed by a force collapse as the peptide lengthened further . Instead, the force level remained approximately constant, whereas length continuously increased. This difference was consistently observed over a range of pulling speeds.Simulations of the SAH domain in the absence of force showed that it maintained a long helical structure throughout the lifetime (200 ns) of the simulations  pairs is disrupted, although the salt bridges are fairly mobile and can break and reform without a break in the hydrogen bond.97) quickly lost its helical structure (within 7 ns) to form a helical bundle, the precise structure of which differed for each simulation . The absence of specific charge-charge interactions between residues as found in the SAH domain means that a single Ala97 helix does not remain stable over long periods of time. In the single molecule force-extension simulations, there was very little increase in force as the length increased . Gly97 rapidly lost its helical starting structure to form a collapsed random coil structure under zero force conditions , which is expected because glycines are not usually part of helical structures and favor coil formation. In force-extension simulations starting from collapsed random coil structures, Gly97 unfolded non-cooperatively at low forces  as expected for a random coil , consistent with our experimental findings. For example, the maximum force values measured at a velocity of 1 nm/ns were 2.3-fold higher for I27 than for the SAH domain. In addition, the forces required to unfold the SAH were higher than those required to extend Ala97 and Gly97 (F and H), demonstrating that the unfolding of the SAH domain is different from that for a random coil (Gly97) or an uncharged helix (Ala97) and that it shows a higher resistance to extension.The behavior of the I27 domain in our modeling nicely matches that observed experimentally in single molecule force spectroscopy experiments. Moreover, the modeling of the SAH domain under zero force conditions shows that this peptide remains helical as observed experimentally by CD, thereby providing validation for the simulation method. At each velocity used, the force required to unfold I27 was consistently greater than the force required to unfold the SAH domain , B and Dnd Gly97 , F and HThese simulations also demonstrate that the unfolding of the SAH domain is non-cooperative in nature in that after the initial elongation phase the force remained approximately constant as the peptide continued to be extended. This type of behavior is reminiscent of that predicted for a phase transition , which oA) with a distance root mean square deviation <0.5 nm on returning to its initial end-to-end distance after unfolding to a partial helix  . Similarly, it was able to refold after rapid extension at 10 nm/ns (B) to a completely non-helical chain (∼30 nm) . In contrast, under the same conditions, I27 does not refold correctly on the same time scale (C). Of note is that for I27 the steps observed represent unfolding events (D).Given the good agreement between simulations and experiments, next the MD simulations were used to predict whether the SAH domain could refold after extension either to a partial helix or a completely extended coil structure as the sensitivity of our single molecule force-extension experiments prevents us from measuring this experimentally for the SAH domain. The simulations showed that the SAH domain was able to refold A with a 10 nm/ns B to a come scale C. Of notg events D.in vitro. It maintains its helical structure when inserted between I27 domains, is effective at separating individual I27 domains, and promotes their refolding after thermal melting. The single molecule force spectroscopy experiments demonstrate that the SAH domain completely unfolds at forces of less than 30 pN at the pulling speed used, and the lack of a characteristic unfolding peak strongly suggests that the peptide is unfolding non-cooperatively. Simulations confirm that the SAH domain unfolds at lower forces than those required to unfold the I27 domain, that it unfolds non-cooperatively, and that uniquely it maintains an approximately constant force during extension. Moreover, the simulations predict that the SAH domain can refold after extension when allowed to shorten back to its original length. These data suggest that the SAH domain does not simply function as a “spacer” separating two functional domains in proteins but has a more complex, force-dependent role.These data show that the 97-residue peptide from myosin-10 that we predicted to be a SAH domain based on our earlier work  behaves e.g. myosin from its binding site on actin). By functioning as a constant force spring instead, the SAH domain allows a variable separation between the domains it connects without increased risk to the integrity of such macromolecular complexes. Although the distribution of charged side chains along the SAH domain is not uniform, the simulations show a near constant force as the SAH domain is extended. The reason for this is that the force measurements in the simulations are a derivative of the free energy. This free energy contains entropy and is not simply a function of the number of salt bridges . Thus, the near constant force we measure in the simulations is derived from the direct estimation of the probability of all microstates compatible with a given extension.The ability of the long α-helix of the SAH domain (27 turns) to maintain an approximately constant force while it is extended suggests that the SAH domain behaves as a constant force spring in which the energy stored is linearly dependent on extension. Importantly, this is different from a Hookean spring in which the force rises linearly with extension, and thus the energy stored has a quadratic dependence on extension. If the SAH domain behaved like a Hookean spring as it is extended the resulting restoring force could quickly rise high enough to detach the SAH-containing protein from its binding partner  domains in the muscle protein myomesin. This helical linker also rapidly and reversibly unfolds at low forces of ∼30 pN , and thiSAH domains are characterized by a high content of charged residues. Our observation that the long predicted SAH region from myosin-10 forms a SAH domain as does the short 37-residue peptide we investigated earlier  indicateThe unfolding behavior and stability of the SAH domain may arise from the frequent charge-charge interactions, which may provide some resilience to stretching, and from the likelihood that salt bridges break individually. When a turn of helix breaks under the effect of thermal fluctuations in SAH domains, it can reform rapidly even if the local structure is considerably deformed. This explains why SAH domains do not show force peaks in AFM force-extension traces in that any of the salt bridges can break in any order. This is in contrast to the unfolding force profile of the I27 domain in which specific hydrogen bonds between the A′ and G β-strands are thought to break first before the rest of the peptide can be extended, giving rise to the characteristic saw tooth unfolding profile .In vivo, in the context of the full-length molecules containing SAH domains inserted between functional domains, this force-length behavior is likely to be important for the cellular role of the protein. How does the SAH contribute to the function of proteins? SAH domains are found in three myosin classes, 6, 7, and 10, which have functions in actin-rich regions of the cell , and ca


Protein domains are commonly used to assess the functional roles and evolutionary relationships of proteins and protein families. Here, we use the Pfam protein family database to examine a set of candidate partial domains. Pfam protein domains are often thought of as evolutionarily indivisible, structurally compact, units from which larger functional proteins are assembled; however, almost 4% of Pfam27 PfamA domains are shorter than 50% of their family model length, suggesting that more than half of the domain is missing at those locations. To better understand the structural nature of partial domains in proteins, we examined 30,961 partial domain regions from 136 domain families contained in a representative subset of PfamA domains (RefProtDom2 or RPD2).We characterized three types of apparent partial domains: split domains, bounded partials, and unbounded partials. We find that bounded partial domains are over-represented in eukaryotes and in lower quality protein predictions, suggesting that they often result from inaccurate genome assemblies or gene models. We also find that a large percentage of unbounded partial domains produce long alignments, which suggests that their annotation as a partial is an alignment artifact; yet some can be found as partials in other sequence contexts.Partial domains are largely the result of alignment and annotation artifacts and should be viewed with caution. The presence of partial domain annotations in proteins should raise the concern that the prediction of the protein’s gene may be incomplete. In general, protein domains can be considered the structural building blocks of proteins.The online version of this article (doi:10.1186/s13059-015-0656-7) contains supplementary material, which is available to authorized users. The discovery of evolutionarily mobile protein domains in the early 1980s, shortly after the recognition of eukaryotic splicing, revolutionized our understanding of protein structure. Before the discovery of the exon-shuffled domains in the EGF receptor , most prToday, domains are central to our understanding of the structure, evolution, and functional roles of proteins and protein families. Protein domain assignments using Pfam , InterPrSome protein domains have clearly understood functions . For exaThe evolutionary, structural, and functional roles of domains suggest that domains are the indivisible building blocks from which larger modular proteins are built. Thus, we were surprised to find that 5% to 10% of protein domain annotations in the Pfam protein domain database suggest that only a fraction of the domain is present in the protein. These partial protein domains can cause problems with iterative profile-based similarity searches . RestricTo investigate the nature of these partial protein domains, we used the Pfam database, which uses hidden Markov models (HMMs) to scan UniProt protein sequences and classify conserved domain regions . Pfam haTo characterize partial domains in proteins, we examined 136 domain families from Pfam27   – from PWhile Pfam provides a very comprehensive annotation of domains in proteins, it is difficult to present statistics for representative Pfam families because of the wide range of family model lengths , family sizes , and number of sequences containing a particular Pfam family . Thus, we focused on the RPD2 subset of Pfam27. RPD2 Pfam families have at least 100 members, with no more than 5,000 sequences for any domain. RPD2 also requires that the Pfam family have at least 200 match states; distantly related partial domains with shorter model_lengths can be difficult to detect. RPD2 also limits families from Pfam clans. Pfam clans . A domain can appear to be full length or partial for different reasons, depending on the sequences that bound the candidate partial.While 80% of RPD2 domains cover more than 90% of the domain model length Figure , 10% of To understand better the computational and biological processes that might produce partial domain annotations, we divided RPD2 candidate partials into three types Figure  based onMore than half of the candidate <50% partials in RPD2 are parts of longer domain annotations that have been broken into pieces by the local HMM alignment. For example, both yeast ALR2 [UniProt:P43553] and MNR2 [UniProt:P35724] contain <50% partial [Pfam:PF01544] domains, but in both of these cases, the missing part of the domain can be found annotated upstream or downstream . Likewise, in [UniProt:E9GP80], a putative uncharacterized protein from Daphnia pulex, the [Pfam:PF00183] (Hsp90) domain cannot be extended to include the missing 394 match states because a different non-homologous domain  sets the N-terminal boundary of the partial domain.Bounded partials are limited in length by either another domain, or by the end of the protein sequence Figure C. Boundeq value) to identify families that are significantly enriched for eukaryotic bounded partials  of <0.05, and 34 at <0.01. We conclude that many bounded partial domains result from inaccurate gene models that produce incomplete proteins.Bounded partials may be produced by unusual protein sequence predictions, e.g., alternatively spliced isoforms that do not produce functional proteins, or inaccurate genome assemblies. To look at the relationship between bounded partial domains and protein sequence accuracy, we asked whether bounded partials are enriched over non-partial sequences in organisms that contain introns (eukaryotes), and thus might be splicing or assembly artifacts. We examined the 94 RPD2 Pfam27 families with 10 or more bounded 50% partial domains and asked whether the bounded partial-containing proteins in these families were more likely to come from eukaryotes than RPD2 proteins that do not contain partial domains. If bounded partial domains are due to inaccurate gene models, we expect the errors more frequently in eukaryotes. We performed Fisher’s exact test on each of the 94 families, and then calculated the false-discovery rate  in reviewed proteins from SwissProt with an Ensembl gene model. During the Swissprot review process, multiple alternatively spliced transcripts with different accession numbers in the TREMBL division of UniProt are merged into a single accession and labeled as isoforms  alignment, the alignment is short because the protein is short and contains a bounded partial.Excluding split domains, a majority of the candidate partial domains belong to the unbounded partial category Figure D. These −25 < E < 10−6 and 29% to 48% identity), but [UniProt:A5BS21] sometimes produces long alignments with more closely related sequences that are annotated as having partial [Pfam:PF01544] domains . Moreover, sometimes [UniProt:A5BS21] produces short alignments with distantly related proteins with longer [Pfam:PF01544] domains  at least 25% of the homolog alignments were <50% of the family model length. These Pfam families are counted as putative partials in Table To identify candidate true partial domains from the set of unbounded partials Figure D, we genAbout one quarter of our candidate partials map to the Pfam model in a way that leaves room for a much more complete Pfam domain /[UniProt:F0YNJ0] vs [PDB:1A6D |B], the Pfam domain aligns with portions of structures with multiple CATH domains, some of which are discontinuous in sequence.In addition to structural partials that could be identified by comparing Pfam and CATH domain annotations, we examined the most abundant putative partials, including [Pfam:PF00852] , about 10% of the domains meet the putative partial criterion. Its closest homolog in PDB is 2ZXC, where it aligns with the N-terminal third of the protein, an 734 , the domain appears to align with repeats of the same structural domain.We compared our hierarchical strategy for identifying evolutionarily mobile structural partials – identification of putative partial Pfam domains followed by confirmation using CATH – with the simpler strategy of looking at CATH domain content in the protein structures annotated by Pfam. Of the 136 Pfam families in RPD2, 107 are mapped in Pfam to PDB structures that are annotated by CATH. We found 64 of those Pfam families map to PDB structures with only one CATH domain, so they cannot be examples of structural partials. In total, 43 RPD2 Pfam families map to PDB structures containing two or more different CATH domains; these Pfam families might be composed of evolutionarily mobile, structurally compact, sub-domains. But only 11 of those 43 Pfam families were confirmed to be both evolutionarily mobile and structurally compact. In 15 cases, either the CATH sub-domain was not consistent with the evolutionarily mobile sequence, or there was inconsistency between the CATH and the VAST annotation. For another 17 Pfam families, although CATH annotates multiple structural domains, we found very little evidence for evolutionary mobility .Our PDB/CATH/VAST+ searches found 11 Pfam families comprising shorter CATH and VAST+ structural domains, and another 7 Pfam families that do not have homologs annotated in CATH, but contain multiple VAST+ domains . RPD2 protein domains are representative of Pfam27 since they have a distribution of partial domain annotation lengths that is almost indistinguishable from all Pfam27 domains with model lengths greater than 200 match states , where 77.8% of the domain annotations are partial, but 72.5% of those partials are split domains. The family-specific nature of partial domains is illustrated by the difference between the extremes  on human trypsin-1 ([UniprotKB:P07477]), while Pfam annotates a single trypsin domain. Since no proteins contain a single CATH half-domain, we would not consider it a structural partial, and we have excluded proteins with repeated structural domains from the list of candidate structural partials in Table .40.10.10We emphasize that these are conservative estimates. Our 18 examples of structural partials excluded cases where CATH and VAST+ disagreed, and other cases where the partial domain topologies annotated on the structure were intermingled because different parts of the same structural domain were assigned to non-contiguous regions of the sequence domain annotated by Pfam. However, because the VAST+ structural annotations do not include the domain-based homology classification provided by CATH, we have less confidence that VAST+ domains can be found in different structural contexts. We are very confident, however, that most of the candidate partials, and about half of the putative partials, are not genuine structural partials. They are more likely the result of sequence annotation and assembly errors.In a companion paper, Prakash and Bateman describe a second mechanism that can produce partial domains in proteins: domain atrophy . PrakashWe suggest that genuine structural partials can be inferred based on two criteria: (1) evolutionary mobility, where the same domain is found in different protein contexts, and (2) structural compactness. However, all structurally compact domains are probably not evolutionarily mobile and there are examples of evolutionarily mobile domains that are not structurally compact. Moreover, in the absence of clear structural data, it is difficult to know whether a domain is in a novel protein context, because even the most sensitive sequence-based comparisons can fail to detect structural homologs. The novel-context question is muddied further by the possibility that some protein sequences were inferred from misassembled genes and genomes. This study shows that most of the candidates we initially characterized as partials are artifacts of partial alignment, splice isoforms, and incorrectly assembled proteins.Accurate domain identification and boundary characterization can dramatically improve protein annotation ,5. IncomThe concept of proteins built from conserved domain building blocks has fundamentally transformed our understanding of protein evolution, folding, and function. However, our ability to identify and accurately bound domain building blocks is hampered by the technical problems inherent in identifying distant homologs and by inaccurate protein sets. A mixture of model-based (HMM and PSSM) and pairwise-alignment methods can provide more reliable domain identification. Our results suggest that partial protein domains should be viewed with suspicion; most protein domains appear to have a characteristic length.Pfam family models, protein sequences, and mappings of curated pfamA domains to protein sequences and domain models were obtained from the MySQL tables distributed with Pfam version 27 , withoutFor this study, the RPD2 families and sequences were projected onto the current Pfam27 domain set, causing some Pfam26 sequences no longer in UniProt  to be drDomain boundaries in the RPD2 protein set were assigned using the pfamA_reg_full_significant table in the Pfam27 mySQL distribution, using seq_start and seq_end to determine the coordinates in the protein sequence, and model_start and model_end to determine the mapping of the protein sequence to the domain model. Only pfamA_reg_full_significant domains with the in_full flag set were used in the analysis, and only sequences with the is_fragment field set to zero were used.The model_length field from the PfamA table was used to determine the full domain length. Partial domains that covered less than 50% of the model_length were identified using the relationship model_end −model_start + 1 <0.5model_length. The number of partial domains changes very little if seq_end and seq_start are used in place of model_end and model_start.Candidate extended domains  were compared to the RPD2 protein sequence database using the SSEARCH program (version 36.3.6) , using tCandidate partial domains were compared to the PDB database of sequences with known structures , using tDrosophila) and identified the subset of RPD2 proteins that have an Ensembl gene model [In addition to identifying sequences with partial domains, RPD2 sequences that lacked <50% partial domains were identified. These sequences, and sequences from RPD2 families with more than ten end-bound partials, were divided into eukaryotic and non-eukaryotic groups based on their National Center for Biotechnology Information (NCBI) taxon_id. Eukaryotic sequence enrichment was calculated using the fisher.test function from the R statistical analysis package. The R qvalue function was used to estimate the number of families that were significantly enriched for eukaryotic end-bounded partials. We also used the NCBI taxon_id to identify proteins from very well-annotated genomes (human, mouse, and ne model .


UBE3A gene and its surrounding chromosome regions has been identified as the causative factor for AS. The human UBE3A gene is located within human chromosome 15q11-13 and encodes an E3 ubiquitin-protein ligase . Due to genetic imprinting of the paternal copy of UBE3A gene in many brain regions, loss of function of a single maternal copy of UBE3A is highly penetrant and pathogenic. Most of the deficits seen in AS patients have been reproduced in Ube3a gene maternal deficiency mice , thus enabling mechanistic interrogations of AS pathogenesis and therapeutic explorations using mice models. Here we briefly discuss recent advances on AS etiology and identify some challenges in translating mechanistic insights into potential therapeutic interventions. Experimental evidence collected so far indicate impaired maternal UBE3A in neurons may contribute to AS deficiency by influencing multifaceted neural developmental processes including cell survival, synaptic transmission, signal transduction, gene expressions.This review briefly discusses key recent research literature on Angelman Syndrome (AS), a rare genetic disorder of neurodevelopmental origin. Dysfunction/inactivation of the maternal Angelman Syndrome (AS) is first reported and named thereafter by pediatrician Harry Angelman in 1965 ,3. Bone UBE3A gene function was identified as the cause of AS by two research groups in 1997 [UBE3A gene encodes the E3 uniquitin ligase UBE3A. UBE3A gene is normally expressed in neurons only from the maternally inherited allele, while the paternal allele is silenced by epigenetic mechanisms known as imprinting. Therefore, mutation of the single maternal UBE3A allele in neurons leads to near complete loss-of-function of UBE3A gene. In the majority of AS patients, UBE3A gene is found inactivated by either intragenic mutation, chromosomal micro deletion in the 15q11-13 regions, paternal uniparental disomy (UPD), or a defective imprinting center (IC) that controls UBE3A transcription [Loss of  in 1997 ,12. The Ube3a gene in mice [UBE3A protein in AS; upon inheritance of the mutation through the maternal germline, Ube3a mutant mice  display salient pathological features of AS. Critical defects in both morphology and function of neurons was found [UBE3A causes AS, increased UBE3A gene dosage  receptors-dependent LTD was potentiated in the hippocampus in AS mice [Intriguingly, experience-dependent maturation of excitatory cortical circuits, and visual cortex function associated with ocular dominance plasticity were found impaired in AS mice, suggesting ysiology . The sam release . Consist AS mice . It has  AS mice . AS mice AS mice  that is  AS mice .A number of combined genetic and molecular studies have shed light on AS etiology. The UBE3A protein, first identified as the mediator of human papillomavirus types 16 and 18 E6 protein ,31, reguUBE3A gene (located in chromosome 15q11–13) [UBE3A gene is likely through paternal expression of a large antisense RNA transcript of UBE3A (UBE3A-ATS) and snoRNAs  in neurons [Ube3a gene, are expressed in a cell-type specific way in the brain. Neurons express maternal sense and paternal antisense, whereas glia express biallelical sense [Ube3a gene resulted in an increase of paternal Ube3a-ATS in AS mouse model [Ube3a-ATS illustrated the inhibitory effect on the expression of paternal UBE3A gene [UBE3A in AS patients is related to ubiquitin ligase instead of to its functional coactivator of transcription of the nuclear hormone receptor superfamily, such as the progesterone receptor (PR) [Ube3a is decreased in mouse neurons after the first postnatal week during which these neurons are undergoing rapid maturation. At the same time, the decrease of paternal Ube3a was accompanied by the nuclei accumulation of Ube3a of maternal origin. Interestingly, in contrast to neuron, glia cells (both astrocyte and oligodendrocyte) seem to express Ube3a biallelically [It has been found that various genetic mechanisms cause the loss (deletion or UPD), inactivation or mutations of maternal 5q11–13) , (Table 5q11–13) –39. Sile neurons ,40. It wal sense . Furtherse model . The UbeE3A gene , and is E3A gene . Anothertor (PR) . The distor (PR) . A recenelically .UBE3A leading to AS are poorly understood. Studies using AS mice have provided some mechanistic insights by demonstrating that Ube3a plays a pivotal role in multiple CNS developmental processes, including cell cycle, signal transduction, transcription and synaptic plasticity [Ube3a expression can influence the viability of neurons. It was reported that the post mitotic neonatal neurons are decreased after maternal Ube3a inactivation in AS mouse hippocampus [Ube3a [The detailed mechanisms on how deficiency of asticity . One pospocampus . The lospocampus , or bothpocampus . Anothers [Ube3a .Ube3a dysfunction. A recent study showed that the Golgi apparatus (GA) cistern was swollen and disorganized in Ube3a maternal deficiency mice, and the pH in GA lumen is increased in cortical neurons. This has implication that a less acidified GA would result in impaired protein sialylation and secretion mechanisms [Ube3a regulate the degradation and turnover of RhoA-GEF Ephexin-5, activity-regulated cytoskeleton-associated protein (Arc), p53, and p27 via ubiquitination [Ube3a function was disturbed in neurons [Another potential mechanism is that protein synthesis including receptors expression can be affected by chanisms . Previoutination ,52,53. Itination . It is n neurons ,55.Ube3a abnormality disturbs the regulation of gene expression. A recent study demonstrated that levels of both Ring1B, which ubiquinates nucleosomal histone H2A to regulate gene expression, and histone H2A, are elevated in many tissues in Ube3a knockout mice [ are confirmed of relevant with AS phenotype [Ube3a leads to changes in neuronal excitability likely through altered membrane biophysical properties.Another hypothetic cause is that out mice . One rechenotype . It was henotype . Anotherhenotype . This stUBE3A in the brain, by retrieval either maternal or paternal copy of the gene. For restoration of maternal Ube3a, one study used recombinant adeno-associated virus (rAAV) to introduce type 2 terminal repeat (TR2) flanked Ube3a into the hippocampus of adult AS mice. The study found that rAAV restored the level of Ube3a in AS hippocampus, rescued the impaired of LTP, and enhanced the cognitive learning as evaluated by Morris Water Maze test [Ube3a function per se, and restoration of Ube3a expression could potentially overcome certain aspects of developmental deficits.The current efforts in therapeutic exploration for AS have been taken on identified putative pathological basis. A conspicuous idea would be to restore the function of the aze test . These rUBE3A allele has the intriguing implication that activation of the silent allele may be able to fulfill the functions of the missing maternal ones, in a way analogous to rescuing neural deficits in adult Mecp2 knockout mice by reinstating the functional Mecp2 gene [Ube3a-ATS expression both in vivo and in vitro could elevate expression of paternal Ube3a [Ube3a could be achieved by blocking the paternal Ube3a-ATS with poly-adenine cassette insertion in AS mouse models. Many resulting AS deficiencies, such as impaired LTP, cognitive deficits, and motor dysfunction were ameliorated [Ube3a expression through non-genetic approaches also seems to hold great promises. Through chemical library screening, Huang et al. (2012) have found that several topoisomerase inhibitors, such as topotecan and irinotecan could resuscitate paternal Ube3a and rescue cellular function in neurons [UBE3A gene in brain can be achieved through bypassing the genetic manipulations.The existence of the intact paternal cp2 gene . In an eal Ube3a . The actliorated . Restori neurons . AlthougUbe3a deficiency in mice is known to impair synaptic transmission and interfere with a critical molecular player in synaptic plasticity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) [Maternal (CaMKII) ,23. Weeb(CaMKII) , the sam(CaMKII) . Cao et Despite these emerging successes in restore neural functions in AS mouse models, outstanding questions and challenges remain in the field. For example,UBE3A dosage is more represented in autism spectrum disorders [What is the definite role of UBE3A in neural connections or circuits within and between many brain regions, and in what molecular context is UBE3A involved to regulate synaptic development, transmission, and plasticity? Why increased isorders ,17 ?How does UBE3A differentially affect both excitatory and inhibitory synapses, favoring an enhanced local circuit hyper-excitability ,25?UBE3A deficiency lead to AS remain enigmatic. The protein substrates of UBE3A in neurons remain to be identified [neurons. Revealing more neuronal molecular substrates or interactomes and how deficiency of maternal UBE3A disrupts cellular homeostasis can be illuminating for AS pathogenesis and molecular intervations.The molecular mechanisms by which entified . Arc, SaUBE3A dysfunction. For example, other genes such as GABAA receptor β3 subunit (GABRB3) gene which locates within the chromosome 15q11-13 locus have been proved playing a role in the AS development. The impaired expression of GABRB3 can render featured phenotypes of AS in mice. These results raised the questions of the definite role of the GABRB3 and its relation to UBE3A in AS genesis [The dramatic variations of symptoms among AS patients imply the contributions of other elusive and perhaps much more complex causes other than maternal  genesis .Some discrepancies exist between AS model mouse behavior and AS patient clinical features. AS mice showed normal social seeking and activity level in contrast to the frequently observed behavioral deficits of AS patients . This maUBE3A expression and correcting AS pathophysiology awaits further experimental validation and extrapolation.On the forefront of AS therapeutic endeavors, the potential of topoisomerase inhibitors in restoring


Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. We describe crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. We show an unexpected nucleotide-dependent interaction of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM–DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates. BLM that result either in expression of truncated proteins or alterations of key conserved residues within the RecQ helicase domain (Bloom's syndrome (BS) is an extremely rare autosomal recessive disorder that is characterized by shortness of stature, a distinctive skin rash and the predisposition to the development of a wide spectrum of cancers at an early age . Individe domain –3. On a e domain , variouse domain  and an ie domain .Bloom's syndrome helicase (BLM), like all RecQ-family helicases, acts as a 3′ to 5′ helicase  on a widThe BLM protein is a 1417 amino acid, 159-kDa polypeptide with multiple structural domains Figure . At its 2+ binding subdomain  and a Winged Helix (WH) DNA binding domain (residues 1069–1192). Two of the β-strands in the WH domain form a prominent hairpin that has been suggested to act as a DNA strand separation pin in other RecQ helicases  could be detected in one study for the BLM HRDC domain  and a single domain llama antibody fragment (nanobody) specifically raised to BLM as an aid to crystallization. We also present two structures of BLM in complex with DNA at 4.7–3.5-Å resolution containing 12-bp duplex DNA with single-stranded 3′-overhangs. Comparisons to other RecQ helicase structures reveals the highly dynamic nature of the protein. Finally, we show that the HRDC domain packs tightly against the cleft formed between the two RecA-like domains D1 and D2 and that this binding is sensitive to the nucleotide status of the enzyme.Determining the ways in which the structural domains of BLM relate and interact with each other will be the key to understanding the role of BLM in the maintenance of genome stability and how it is able to catalyze complex reactions such as double Holliday junction dissolution. In this study we present the 2.8-Å resolution crystal structure of BLMEscherichia coli and purified at the SGC protein production pipeline . In summary, 94 purified antigens, including eight membrane proteins, were collected from the Structural Genomics Consortium. All proteins were expressed in pipeline . Two nonE. coli with a PelB leader and a C-terminal His6 tag. The full protein sequence is indicated, with the asterisk indicating the point of cleavage during export to the bacterial periplasm.Nanobody CA5075 was subcloned to be expressed in MKYLLPTAAAGLLLLAAQPAMA*QVQLQESGGGLVQAGGSLRLSCAASGIWFSINNMAWYRQTPGKQRERIAIITSAGTTNYVDSVKGRFTISRDDAKNTMYLQMNSLIPEDTAVYYCNLVADYDMGFQSFWGRGTQVTVSSHHHHHH.E. coli grown in Terrific Broth with 0.1-mg/ml ampicillin at 37°C to OD = 3; the temperature was then lowered to 28°C. After 1 h, 0.2-mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and incubation continued overnight at 180 RPM. The cells were collected by centrifugation and suspended in 100-ml HES buffer (200-mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), pH 7.5, 0.5-M sucrose, 0.5-mM ethylenediaminetetraacetic acid (EDTA)) and frozen at −80°C in 50-ml aliquots. Fifty millilitre of cell suspension was thawed and kept on ice for 1 h with occasional mixing. The cells were diluted with 50 ml of water. After another 1-h incubation on ice, the cells were removed by centrifugation and the protein was recovered from the supernatant (periplasmic extract). The protein was then purified by binding to Ni-IDA Sepharose, which was washed and eluted with 0.3-M imidazole then further purified by gel filtration in Superdex S75 in 50-mM HEPES, pH 7.5, 0.5-M NaCl, 5% glycerol.The protein was expressed in 3 l of 636–1298  was expressed in E. coli and purified by Ni-Sepharose, tag cleavage, heparin-Sepharose and Superdex 200 as described in the Supplementary information of  protease and gel filtration on Superdex S75. The mutants H666A and S729A were generated by site-directed mutagenesis and purified using the same method.BLMGU452710 ), which 1206–1298, encoding the HRDC domain, was cloned into pCDF-BirA (Genbank JF914075 (10 tag at the N-terminus and a biotin acceptor sequence at the C-terminus. The protein was expressed in E. coli in TB medium supplemented with 20-mg/l biotin and purified using Ni-IDA affinity capture and gel filtration on S75 Superdex column. The K1270V mutant was similarly purified.BLMJF914075 ), which Saccharomyces cerevisiae-strain JEL1. Cell growth, protein induction and purification were performed as described in (The BLM(S666A) and BLMS729A) variants were generated from pJK1-BLM(WT) by a site-directed mutagenesis strategy. The resulting pJK1- BLM(S666A) and pJK1-BLM(S729A) were transformed in 29A varia4 were added to 0.5 mM each. The proteins were then concentrated to 17 mg/ml and crystallization was set up with standard crystallization screens supplemented with 15% glycerol. Crystals for the BLM Nanobody complex were obtained from conditions comprising 0.1-M MES pH 6.0, 22% PEG 20000 and glycerol and were cryoprotected by soaking in a solution consisting of the well solution supplemented with 15% glycerol. Crystals were loop mounted and plunged into a pool of liquid nitrogen. Diffraction data extending to 2.8-Å resolution were collected on beamline I24 at Diamond light source. The diffraction data were processed with the program XDS (Rfactor of 20.4% (Rfree = 24.7%), with no residues in the outlying regions of the Ramachandran plot.BLM protein precipitated in low-salt buffers unless supplemented with glycerol or other stabilizing additives. For co-crystallization with nanobodies, the BLM and nanobodies were mixed in GF buffer  and the complexes were purified by gel filtration (Superdex 200) in the same buffer. ADP and MgSOgram XDS  and belogram XDS  and the gram XDS  and a na2+ added at a final concentration of 1 mM in a buffer containing 4-mM HEPES, 200-mM NaCl, 16.7% glycerol. Crystals of the BLM DNA complex I were obtained from conditions comprising 21% PEG 4000, 0.1-M HEPES pH 7.6, 5% isopropanol. Crystals of BLM-DNA complex II were obtained from well solutions comprising 19% PEG 4000, 0.1-M Hepes pH 7.2, 5% isopropanol. Both crystals were cryoprotected by soaking in a solution consisting of the well solution supplemented with 23% ethylene glycol, before being loop mounted and plunged into a pool of liquid nitrogen. Data sets extending to ∼3.2-Å resolution (complex I) and 4.8-Å resolution (complex II) were collected on beamline I24 at Diamond light source. Data were processed with XDS , and using a rigid body refinement strategy for complex II . In both cases it was noted that a significant improvement of the electron density maps was obtained by B-factor sharpening, presumably a result of the large Wilson B factor and consequently high B-factors in the models which down weight the high-resolution terms in the map calculation. B-factor sharpening was implemented using a sharpening B-factor determined automatically by phenix refine. A full summary of the data processing and refinement statistics is shown in Supplementary Table S1.Preparation of oligonucleotide duplexes was performed as outlined above. DNA complexes were prepared by mixing BLM (15 mg/ml) and DNA in a molecular ratio of 1:1.1 with ADP/Mgwith XDS , and thewith XDS  and the with XDS , using t2, 90-mM NaCl and 200-μg/ml BSA. Reactions were incubated 15 min at 37°C and stopped by the addition of 0.2% sodium dodecyl sulphate, 10-mM EDTA and 100-μg/ml Proteinase K. After 30 min of incubation at 37°C, reaction products were separated with 12% acrylamide gel at 15 V/cm constant for 1 h at room temperature. Gels were dried and exposed overnight on a phosphor-imaging screen. The image was recorded using a Typhoon scanner.The fork and 4-way junction substrates were prepared as described in . One femdHJ substrate was prepared as described in . Reactio2, 1-mM DTT, at 37°C for 10 min. The reaction mixtures contained 0.5-mM ATP, 500-nM single-stranded DNA (50-mer oligonucleotide) and 1–100-nM BLM. The reactions were stopped, and the amount of hydrolyzed ATP was monitored by the malachite green method, using a commercial kit (Innova Biosciences).ATPase activity was measured in 50-mM Tris pH 7.5, 50-mM NaCl, 5-mM MgClTo check the ability of two mutants to correct SCE frequency in BS cells; GM08505 cells, an SV40-transformed fibroblast cell line from a BS patient, were transfected with wt or mutant forms of BLM cDNA in pcDNA3 plasmid, as described previously . The celBinding kinetics of RecA domains to HRDC domain were determined by the biolayer interferometry (BLI) method  using thAfter reference correction, the subtracted signals from HRDC-coated biosensors were analysed in the FortéBio analysis software provided with the instrument.www.BIOSIS.NET). Rigid body modelling was performed using the program SASREF  functions were calculated using GNOM . Scatterm SASREF .Despite repeated attempts, we were unable to crystallize BLM in the absence of stabilizing agents. For this reason we used BLM-specific nanobodies as an aid to crystallization . In para636–1298-ADP-nanobody complex diffracted to 2.8-Å resolution, and the structure was solved by molecular replacement. Examination of the crystallographic contacts provides a rationale for the role of the nanobody in promoting crystallization, with specific inter- and intra-molecular contacts mediating crystal packing . The overall architecture of BLM is a large globular core, formed primarily by the two RecA domains, to which the Zn2+ binding and HRDC domains are closely associated . The structures of the individual domains of BLM are virtually identical between the nanobody and DNA complexes . However, the relative orientation of the WH domains is markedly different, with a rotation of 90° required to place the domains in equivalent positions. The WH domain in the DNA complex structure is in a similar position to that observed for human RECQ1 HsRECQ1 ,53) and, and,53))E. coli RecQ (EcRecQ) structures  across the three RecQ family structures, differing mainly in the length of the helical hairpin (BLM > HsRECQ1 > EcRecQ).The Znibid).As noted previously ,24,58, tIn order to gain insight into the mode of BLM DNA binding we have determined the crystal structure of BLM, in the absence of a nanobody, but in complex with ADP and two different DNA oligonucleotides, containing 12 bp of duplex DNA with either a 5 or a 12 nucleotide 3′ overhang. The best diffraction (3.5-Å resolution) was obtained for BLM DNA crystals with the 5 nucleotide overhang (complex I); crystals containing a 12 nucleotide overhang (complex II) diffracted less well (4.5-Å resolution).2+ binding domain and the WH domain , the duplex region of which is positioned between the D2 domain, the Znn Figure  and D.Consistent with a role in strand separation ,23, the 868PKKPKK873) that connects the first β-strand to the first α-helix of the D2 domain. Two residues at the tip of this loop K870 and K872 form salt bridges with the phosphodiester backbone. HsRECQ1 lacks these residues and instead contains a longer loop that would make steric clashes with the DNA if similarly positioned. A summary of protein–DNA contacts is depicted in Figure A strand separating hairpin is a common feature of DNA helicases of superfamily 2  as well as SF1 (e.g. PcrA). However, the overall geometry of the interaction with DNA differs markedly . In BLM the DNA duplex is almost perpendicular to the 3′ overhang, whilst in the HsRECQ1 DNA complex it is almost parallel. HsRECQ1 resembles HEL308, whilst BLM is more similar to the WRN WH-DNA complex ; not sho. The pos2+ binding domain to contact the D2 domain , that are appropriately spaced (∼7.5 Å) to correspond to the positions of the sixth and seventh phosphates of the 3′ overhang . Due to the low resolution of this data set, only phosphate positions have been modelled for these two bases, and contacts to these phosphates are inferred from the proximity of the respective groups rather than directly observed in the electron density. The phosphate of the sixth base is in an intermediate position between the D2 and D1 domains and does not interact with the rest of the protein. In contrast, the seventh base of the 3′ overhang is closely associated with the D1 domain, close to the conserved helicase motif Ia. The side chains of R717 (part of motif Ia) and R808 (part of the conserved ‘aromatic-rich’ loop) are sufficiently close to provide favourable electrostatic interactions with the phosphate oxygens Figure , and mutThe HRDC domain has been found to be dispensable for the core helicase activity of BLM . The HRDin vitro and in cells. Based on the reported functional deficiencies of the K1270V mutant, which lies in the HRDC–D1 interface, we also generated two mutants in the D1 domain (H666A and S729A) that are also part of this interface, and tested their functional consequences.To examine the validity of the HRDC-core interaction and its implications on the activity of BLM, we performed binding experiments with the isolated domains, as well as small-angle X-ray scattering (SAXS), and functional analysis To measure directly the interaction between the HRDC and RecA domains in solution, we measured the kinetics of this association using individually expressed domains by Biolayer interferometry. Despite problems associated with significant non-specific binding to the sensor tip, we were able to confirm an interaction between the HRDC domain and the RecA core in the presence of either ADP or ATP with an apparent affinity of 30-100 μM Figure , suggest636–1298 in the presence and absence of the nucleotides ADP, ATP and the ATP analogue ATPγS , as well as the overall shape, are in good agreement  , suggesting the conformation BLM in the Nanobody crystals is a reasonable approximation of the nucleotide-bound DNA free structure. The flexibility of the various domains may also play a significant role in the scattering in solution, and analysis of the various experimental curves by the Porod–Debye law  as well as in helicase and dissolution efficiency. The H666A and S729A mutants appear to be more defective (up to 3-fold) when assayed for 4-way junction branch migration. Finally, the ability of all three mutants to correct the enhanced SCE frequency of BS cells in vivo was very similar to that of WT BLM . This s2+ is bound to the expected position of the Mg2+ ion. In addition, residues 798–808, which form the aromatic-rich loop, are disordered in that structure (4O3M) and the helical region of helicase motif V adopts a significantly different conformation. These differences are particularly significant given that these structural motifs have been found to play a role in energy coupling  have catalytic cores with high sequence and structural similarity, yet their physiological roles hibitors ,68. In thibitors  of a ratE. coli RecQ ; this binding is dependent on the presence of nucleotide (ADP or ATP), and is disrupted by mutations in residues lying in the interface in the crystal structure.636–1298, which shows significant conformational changes associated with nucleotide binding and release. These changes are consistent with theoretical calculations of the scattering curves from models in which the HRDC domain is associating and dissociating from its binding cleft between the D1 and D2 domains. These findings are consistent with two roles of the intramolecular interaction . First, the HRDC domain may influence the mechanochemical coupling of the ATPase cycle, perhaps playing a role in ensuring the correct relative positioning the single-stranded DNA binding regions of the D1 and D2 domains. Alternatively, the cycle of association and dissociation of the HRDC domain with nucleotide binding and release may control a more global aspect of the catalytic cycle. An intriguing possibility relates to the extended loop connecting the HRDC and WH domains which protrudes from the rest of the BLM surface, creating a tunnel-like structure when the HRDC is bound. The proximity of the blunt end of the DNA duplex to this loop leads us to suggest that the threading of ssDNA through this loop and its subsequent association with the HRDC domain may be a feature of BLM activity on complex substrates (in the context of the dissolvasome) such as during the late stages of the dissolution process .Further evidence for a role of the HRDC domain in conformational changes comes from our SAXS analysis of BLMWe have attempted to investigate the role of the HRDC–D1/D2 interaction in the biochemical and cellular activities of BLM by generating structure-guided mutations affecting the binding interface. We were prompted by a previous study, identifying the K1270V HRDC mutation, which affected double Holliday junction dissolution activity . Two resVmax for DNA unwinding, higher Km for DNA and a generally less efficient coupling of ATP hydrolysis to DNA unwi6nding (Further evidence for a role of the HRDC domain in the catalytic function of BLM comes from a recent structural and biochemical study in which an enzymatic characterization of BLM mutants lacking the entire HRDC domain was performed . In genewi6nding . This stPDB IDs: 4CGZ and 4CDG.Supplementary Data are available at NAR Online.


Encyclopedias are not a new platform for the distribution of knowledge, but they have recently drawn a great deal of attention in their online iteration. Peer production in particular has emerged as a new mode of providing information with value and offering competitive advantage in information production.Large numbers of volunteers actively share their knowledge by continuously editing articles in Baidu encyclopedias. Most articles in the online communities are the cumulative and integrated products of the contributions of many coauthors.Email-based surveys and objective data mining were here used to collect analytical data. Critical mass theory is here used to analyze the characteristics of these collective actions and to explain the emergence and sustainability of these actions in the Baidu Encyclopedia communities.These results show that, based on the collective action framework, the contributors group satisfied the two key characteristics that ensure the collective action of knowledge contribution will both take place and become self-sustaining. This analysis not only facilitates the identification of collective actions related to individuals sharing knowledge in virtual communities, but also can provide an insight for other similar virtual communities’ management and development. Peer production is a new mode of production that has recently emerged from software and content production. This mode, which is based on sharing and cooperation, has spawned almost all of the mature operating system such as GNU/Linux and various BSD systems and innumerable other free software applications. The advent of so-called Web 2.0 technology has infused this mode with new vigor. One of the typical cases of peer production based on Web 2.0 technology is the Online Encyclopedia.With the development of Web 2.0, Internet users ceased to be solely passive recipients of information. They can now share their own knowledge and interact with others in collaborative workspaces  involves decreasing marginal returns to contributions and refers to situations in which the first few units of resources contributed have the largest effect on the collective good and subsequent contributions progressively less. In contrast, accelerating production function  involves increasing marginal returns and causes subsequent contributions to generate progressively larger payoffs. In this way, each contribution makes the next one more likely. Noticeably, the collective good with accelerating production function will suffer the daunting start-up problems but optimization and sustainability can be achieved once the initial contributions are obtained.Group heterogeneity is favorable to collective action. The significance of this factor has been widely recognized since the emergence of critical mass theory. As Oliver et al.  pointed http://baike.baidu.com/). Apparently, the Baidu Encyclopedia can make use of the advantages of its position as a late starter.Undoubtedly, Wikipedia is the most extensive and well-known Wiki application. Wikipedia has also attracted growing academic attention due to its popularity and unconventional modes of operation Ashton . Baidu Ehttps://en.wikipedia.org/wiki/ Wikipedia:Administration, Retrieved July 12, 2009). The Baidu Encyclopedia is a Chinese-language collaborative Web-based encyclopedia provided for use with the Chinese search engine Baidu . The Baidu Company applies a Wiki philosophy to the encyclopedia, which is mean to complement the search engine and two other services (Zhidao and Post). All articles written and edited by registered users must be reviewed by behind-the-scenes administrators before publication in order to be self-censored in accordance with the government regulations. However, the Baidu Encyclopedia is an open-Internet encyclopedia. In this capacity, it espouses equality, collaboration, and sharing, and it provides a voluntary environment that allows collaborative contributors and maintenance in a simple and effective manner. Most low-level positions open to all volunteers, while only individual high-level administrative positions are given out by appointment. Any Internet user in the Baidu Encyclopedia can easily modify any article or create a new one.However, Baidu Encyclopedia is more like a combination of a self-organized community and other types of organizations, while the Wikipedia community has been viewed as a self-organized community. Wikipedia is a non-profit online encyclopedia, and it provides people with the freedom to share knowledge Akiyoshi . AlthougAt present, the available languages in the Baidu Encyclopedia include its primary language, simplified Chinese, as well as traditional Chinese, Korean, Vietnamese, and Japanese, which are automatically translated into simplified Chinese. All registered users’ contributions are evaluated and rewarded using a credit point system. There are two forms of assessment credits in the Baidu Encyclopedia: experience value and wealth value. Experience scores are based on what someone does, regardless of the results, and wealth scores are based primarily on results. Experience scores also relate to the level and honor within the community. Each Baidu Encyclopedia user can create a personal space in which he or she can share historical contribution information, personal characteristic information, and the honors and rewards offered by Baidu Encyclopedia. In this way, the Baidu Encyclopedia can provide large number of objective analytical data that can be used to analyze the collective knowledge sharing actions within this virtual community.The study was reviewed and approved by an institutional review board Zhongshan Institute, University of Electronic Science and Technology of China. (ethics committee)Are the contributors’ interests heterogeneous?Are these contributors’ ability to provide resources heterogeneous?What is the type of production function?Critical mass theory provides a complex theoretical model of the production of collective action and it can be used to explain the emergence and sustainability of collective action. There are three research questions that must be answered when using critical mass theory to examine the emergence and predict the sustainability of collective action concerning knowledge sharing in virtual communities:As described above, the Baidu Encyclopedia and Wikipedia have both survived the start-up problem and currently provide a fruitful crop of analytical data that can be used to answer the research questions.Generally, most editing projects in Baidu Encyclopedia are based on common areas of knowledge or interest such as hobbies, industries, popular culture, and geographic areas. Noticeably, knowledge sharing on the encyclopedia takes time and effort, and contributors rarely derive explicit material benefits other than virtual rewards and prestige among their peers. Many prior studies have preferred to use motivational theory to interpret individual knowledge-sharing behavior and confirm that motivation has a key role in the intent and behavior of knowledge sharing  An email-based survey was performed to assess motivation; and (2) data mining was used to mine available objective data in specific sub-communities to assess individuals’ educational attainment and career distribution.Most previous studies have shown that motivation underlying knowledge sharing comes primarily from a desire for personal growth, operational autonomy Tampoe , a senseTo assess the motivation of participants in Baidu Encyclopedia, we have to contact large numbers of contributors to carry out the survey. Fortunately, Baidu Encyclopedia allows a register member to contact the contributors in a form of private letter within the platform. However, many contributors would not reply the private letters. We randomly and continuously sent lots of private letters to explain our motivation of the survey. Finally, we received two hundred contributors’ replies. Hence, two hundred contributors were randomly chosen and an online survey questionnaire was conducted through interactive media, such as QQ, e-mail. The items used to assess motivation were mainly adapted from previous studied , the category navigation system can help searches for different categories. As of June 18, 2013, there were 6,235,088 articles produced by 3,249,307 contributors. Data regarding the revision history of articles in the “physics” class and the “mathematics” class were here mined. The mined data are given as “Article-Edited Time-User.” 28,267 available revision history data are mined, including 13,144 available revision history data in the “mathematics” class and 15,483 available revision history data in the “physics” class  was collected from their personal space.Prasarnphanich and Wagner  ever exaFigure Since the quality of most is indicated by the number of edits made Lih . Here, tAlthough the actual scatter fluctuated wildly over time, the fitted curve indicates a pattern of accelerating production function, which coincides with the description in the reward score system. This accelerating production function can draw the interest of subsequent contributors, which can draw even more subsequent contributors to take part into the editing process.One interesting part of critical mass theory is that collective action usually requires reaching critical mass—a small segment of the population that chooses to make big contributions to the collective action while the majority does little or nothing. These small segments that make large contributions to collective action are the critical mass for that the collective action. Note that, in the Baidu Encyclopedia community, each user’s number of edits in sub-community can be viewed as the local contribution while the wealth value can be viewed as the global contribution. In this way, the distributions of these users’ wealth value and number of edits can be used to describe this phenomenon as follows Fig. :Fig. 6DiThe histograms of wealth value and number of edits indicates that 97.96 % contributors gather were concentrated at or near the low endpoint of the wealth value spectrum and 99.97 % of contributors were concentrated at or near the small endpoint by number of edits made. These results prove that most contributors (97.96 %/99.97 %) make little or no contribution to the articles while only small segment of contributors (2.04 %/0.03 %) make large contributions to these articles. Actually, most people simply edit the articles that they happen to be reading rather than make substantial contributions to these collective actions. Lih  ever argPeer production systems are a new mode of production, and they are increasingly influencing our lives. Online encyclopedia-based Wiki systems play an important role in the transmission and sharing of knowledge. In these virtual communities, large numbers of volunteers transmit and share their knowledge by editing articles. Any Internet user can potentially benefit from these articles. In this way, making and improving these articles can be viewed as public good. Baidu Encyclopedia and Wikipedia provide a simple and easily operate platform for these volunteers to promote collaboration. In this way, the Baidu Encyclopedia has also survived the start-up problem, which is challenging for some collective action.Critical mass theory was here applied to Baidu Encyclopedia to explain the emergence and to predict the sustainability of the knowledge sharing action in virtual communities and to study the crucial factors by which collective action would emerges and becomes self-sustaining. These results show the following: (1) Users of the Baidu Encyclopedia have many different reasons for editing articles, such as enjoyment, altruism, reciprocity, and earning more points. (2) Users’ personal resources, which included a variety of career and educational backgrounds, were significantly different. Most contributors are well-educated and still involved in the educational field, often as students. Knowledge communities should encourage those who are educated or well-informed to edit and evaluate the articles. (3) The scoring rules used to evaluate the user’s contributions and assess each user’s reward and honor shows that the type of production function is accelerating function, but also the production function assessed by the number of contributions versus the number of edits over time also showed an accelerating function. These results were entirely consistent with the argument that a belief in furthering the collective good causes production function to accelerate. These results also indicate that an increasing number of users may take part into the collective workspace. (4) The distribution of users’ local and global contributions also indicated that in these communities, only a small segment of the population chose to make large contributions to the collective action while the majority did little or nothing.Other statistical characteristics of articles, such as the number of edits undergone and the number of contributors involved can also be extracted. These results show that most articles in Baidu Encyclopedia are still in their initial stages. More articles need to be further modified.In summary, the virtual community of the Baidu Encyclopedia can reduce the costs of creating and publishing information, which also reduces barrier to participation. The development of the community mainly involves on the persistent contributions of heterogeneous groups of users with many different motivations and ways of participating. Analyses show that Baidu Encyclopedia meets key conditions that the collective actions of knowledge contribution emerge and become self-sustaining. Increasing numbers of users then actively participate in discussions and creation of content on the Internet.In addition, the insight can be obtained from current analyses for other similar existing communities: It is important to establish mechanisms that may attract different kinds of users, especially educated or well-informed users, and keep them involved. There is no denying that this study has some limitations. First, there may be other factors that can influence the collective action of knowledge sharing among virtual communities. Secondly, only career and level of education was used to explain the resource heterogeneity. Certainly, more factors needed to be evaluated to facilitate analysis and explain heterogeneity. It is currently difficult to directly assess the content production function. In this way, we not only explained the type of production function using the score rule, but also adopted the curve fit to assess the type of production function. However, more reasonable methods of assessing the type of production function are planned.