P < 0.001). During winter, QBA yielded differences between herds over PC1  (P = 0.032), but not PC2 (mood) (P = 0.139). Through summer, there was a strong difference across both PC1 (P < 0.001) and PC2 (P = 0.002), with HG exhibiting more positive behaviour. A difference was found in hair cortisol levels, with the greatest concentrations observed in HG (P = 0.011), however such a pattern was not seen for nasal mucus cortisol, or for serotonin. Overall, providing summer grazing (HG) appeared to afford welfare benefits to the cattle as shown with more positive QBA assessments, but also slightly better health indicators, notwithstanding the higher levels of cortisol in that group.Animal welfare encompasses all aspects of an animal’s life and the interactions between animals. Consequently, welfare must be measured across a variety of factors that consider aspects such as health, behaviour, and mental state. Decisions regarding housing and grazing are central to farm management. In this study, two beef cattle systems and their herds were compared from weaning to slaughter across numerous indicators. One herd (“HH”) were continuously housed, the other (“HG”) were housed only during winter. Inspections of animals were conducted to assess body condition, cleanliness, diarrhoea, hairlessness, nasal discharge, and ocular discharge. Hair and nasal mucus samples were taken for quantification of cortisol and serotonin. Qualitative behaviour assessments (QBA) were also conducted and performance monitored. Physical health indicators were similar between herds with the exception of nasal discharge which was more prevalent in HH ( Animal welfare is an important component of decision making on farms and is something desired by consumers and wider society . Within The demand for ‘ethical’ animal products is increasing, driven by a moral view from consumers about the societal responsibility to care for animals . ConsumeWelfare work on cattle has overwhelmingly focussed on the dairy sector, due to larger herd sizes, greater preference towards continual housing (relative to beef) and the fact that the output (milk) is produced continuously and thus any short-term adversity has an instant financial impact. There is, therefore, a need to improve our holistic understanding of beef cattle welfare. Assessing animal welfare, however, is challenging. Welfare is not one single variable; it is a complex interaction of all aspects of an animal’s existence and being. It is therefore essential to address welfare assessment holistically, considering factors spanning different aspects of the animal .Of the five ‘Domains’ of animal welfare, i.e., Nutrition, Environment, Health, Behaviour and Mental State , the heaThis work aimed to address the question: How do the health and welfare of beef cattle differ between housed systems and conventional systems? For that purpose, we assessed two beef cattle herds, with and without access to pasture after winter housing, across a variety of health, welfare, and performance metrics.Physical health – Body condition and health inspections conducted on an individual animal basis. Also, veterinary records for any treatments.Behavioural – QBA was conducted at a group level.Endocrinology – Concentrations of cortisol and serotonin in both hair and nasal mucus on an individual animal basis.Performance – Analysis of monthly liveweight data.This study compared the welfare of two herds of finishing beef cattle, one in a housed system and the other in a conventional system. Welfare and health were assessed across four key areas: The experiment was conducted at the Biotechnology and Biological Sciences Research Council's National Bioscience Research Infrastructure, hosted at Rothamsted Research: The North Wyke Farm Platform   and stabiliser cross (STX) cattle, all of which derived from the same suckler herd. Animals were allocated from the suckler herd at weaning (27 October 2020) to one of the two systems. At allocation both resultant herds had the same mean age and weight . The sex ratio was similar between herds  as was breed ratio .Two beef rearing systems were compared. The first system had cattle housed all year around, named “HH (housed-housed)– The herd was housed from weaning (27 October 2020) until finishing at ~14-15 months age. During this time, they received grass  silage ad libitum, 4 kg per head of rolled barley (Hordeum vulgare), and 0.3 kg per head of wheat (Triticum aestivum) distiller’s grain.HG (housed-grazing) – This herd were housed from weaning (27 October 2020) through winter, during which they received grass silage  ad libitum. In spring, the herd were turned out onto pasture (13April 2021), which was dominated (>0.60) by perennial ryegrass, with a smaller abundance of creeping bent (Agrostis stolonifera), Yorkshire fog (Holcus lanatus) and marsh foxtail (Alopecurus geniculatus) also contributing to the sward; legumes comprised <0.01 of the overall composition  and concrete floored area (3.5 x 24 m). Straw bedding was added to pens daily using a tractor trailed straw chopper. The concreted area was scraped every morning to remove dung and urine using a tractor mounted yard scraper. The entire daily feed ration was distributed along the length of a ground-level feed passage using a tractor trailed forage mixer wagon every morning after bedding and scraping. The feed passage was adjacent to the concreted area and allowed access to feed through diagonal bars. Every evening the remaining feed was pushed up using a tractor mounted silage pusher to ensure it was able to be reached by the animals. The forage mixer wagon was equipped with weigh cells enabling accurate measurement of rations.Animals were weighed every ~4 weeks and selected for slaughter on the basis of visual assessment and palpation of skeletal reference points as they approach target weights  and fat class . Throughout the course of the study, the number of cattle in the HH herd decreased as they were sold to slaughter as part of normal management practice  this wasCattle were examined across eight health indicators that covered a range of symptoms associated with common disease/infections of cattle. These were selected and adapted from the Welfare Quality Protocol  and AssuQualitative behaviour assessment (QBA) was conducted on a weekly basis for both herds. Assessments were conducted by a single assessor with previous experience conducting QBA on beef cattle and who is a qualified professional in animal science with approximately seven years’ experience. In previous studies  were analysed for cortisol and serotonin concentrations. Cortisol was quantified by enzyme linked immunosorbent assay (ELISA) using a commercially available kit . Methodology was adapted from the manufacturer instructions and are further detailed in Results for physical health indicators were analysed using cumulative link models (CLM). The dependent variable was the score for the relevant indicator and the independent variables were season, timepoint (nested within season) and herd. The exception to this was the measure of ‘hairlessness’ which had insufficient variance of results for CLM analysis, instead, a general mixed effect model (GLMM) was conducted.t-tests were conducted to compare herds across both PC1 and PC2.Results from QBA were subject to PCA analysis with scaling and centring . From this, first and second principal components (PC1 and PC2) were derived. Results were reported with 95% confidence ellipses for each treatment each season. For each season, two-tailed Hormone data was analysed using repeated measures ANOVAs with concentrations as the dependent variable and timepoint and herd as independent variables. Post-hoc Tukey testing was then conducted to determine differences between herds at all timepoints.Cattle growth rates were compared using a general linear model (Gamma distribution) that considered herd, breed, and sex as factors.Across all timepoints and both herds, hormone concentrations, negative health scores, and performance (average daily gain) were compared against each other by way of Kendall’s rank correlation. Negative health scores were determined per animal, per timepoint, as the sum of scores for cleanliness, diarrhoea, hairlessness, nasal discharge, ocular discharge, swelling, lesions, and lameness.Statistical analysis and figures were performed in R (i386 4.1.2)  and R StLameness: no incidences were observed over the study period. Swelling: one swelling was observed on the head/neck of an animal in the HH herd at timepoint Summer 1, which was scored as 2 (≥ 2cm diameter). Lesions: one lesion was observed on the head/neck of an animal in the HH herd at timepoint Summer 1. BCS: scores were significantly greater in the HH herd than HG . During winter, across both herds scores remained relatively stable . Scores were significantly higher in summer than in winter in both herds  and increased significantly over summer . Cleanliness: no significant difference was found between the two herds across the study period . There was a significant difference in cleanliness scores across time  and, notably, during winter cleanliness scores were far worse at the end compared to beginning. Cleanliness appeared to improve over summer with animals significantly cleaner at the end compared to start  and, overall, animals were cleaner in the summer than in the winter . Diarrhoea: scores followed a similar trend to cleanliness scores. Higher (worse) scores were seen in winter compared to summer , scores were worst at the end of winter compared to start  and there was an improvement over summer . At timepoint Summer 2, the diarrhoea rate (at score 2) was higher for the HG herd (43.3%) compared to the HH herd (25.9%). Across the entire study period there was no statistically significant difference between the two herds . Hairlessness: incidences were relatively uncommon, though there was a peak for both herds at winter 2 which was significantly greater than at other time points . There was no significant difference between herds . Nasal discharge: At all timepoints scores were significantly  higher (worse) for the HH herd than for HG. A similar temporal effect to cleanliness and diarrhoea was seen with scores being greatest in winter  with incidence increasing  over winter  and decreasing through summer . Ocular discharge: incidences were uncommon in both groups and thus there was no significant difference between herds  or seasons .Differences were observed in scores for physical health indicators both between herds and across time . LamenesVeterinary treatment records showed that a total of six different conditions were treated for during the study , three ot = 2.23, P = 0.032) but not PC2 . The difference in arousal is represented by marginally calmer outcomes observed for HH relative to HG. During summer there were strong and significant differences between herds across both PC1  and PC2 . Outcomes of HG were associated strongly with constructive and socially cohesive behaviours. There was larger variation in behaviours for HH, but with an inclination towards negative behaviours and boredom.PC1 and PC2 accounted for 0.49 of variance .PC1 was -1 across both winter timepoints to 1.26 and then 0.93 pg mg-1. This yielded a significant difference between herds across the time points . There was also a significant effect of time . Temporal variations in nasal mucus cortisol concentration . However, the difference between herds was not significant across all time points . For the HH herd, concentrations increased from Winter 1 to Summer 1, with a sharp drop off in Summer 2. Whilst a similar shape is seen for the HG herd, it is less extreme and also earlier with a peak in Winter 2 and reduction through the Summer.For the HG herd, hair cortisol concentrations  remainedntration  were sigF = 56.51, P < 0.001). This was characterised by an increase in concentrations over winter followed by a sharp drop off in summer, for both herds. There was no significant difference in concentrations between herds . Concentrations of serotonin within nasal mucus . Across both herds, there appeared to be a gradual decrease over winter which somewhat levelled out moving through the summer. Whilst nasal mucus serotonin concentrations were higher in the HH herd than HG, this was not significant at α = 0.05 .Hair serotonin concentrations  showed sal mucus  varied s-1) compared to HG (0.74 kg day-1)  (-1) than STX (0.83 kg day-1) . Despite steers finishing with a greater liveweights than heifers, they started the study period at a greater weight and thus differences in ADG were not significant .Average daily gain (ADG) was significantly greater for the HH herd (1.21 kg day< 0.001) . A signiP < 0.001) correlation of hair serotonin with negative health. The other significant correlations were weak (τ = 0.180 or less).Metric correlations were statistically significant for 8 of 15 parings . There wpersonal communication, 2021). Regarding the four incidences of mange in the HH herd, the condition may be associated to barn conditions  to growth outside of diet. This was reflected in BCS data as scores for both herds increased over time towards finishing.During winter, QBA outcomes of both herds were similar, reflecting their comparable housing. The dispersal was relatively broad across PC1 and PC2, similar to results by -1) was towards the lower ends of those observed in cattle elsewhere [12.15, . Consequently, the quality of care and resources available for husbandry were high for both herds and the interpretation of results is limited by that context. In other circumstances, risk factors associated with housing or grazing may be present or exacerbated in ways not evident from this study and the likelihood of this may not be equal across both systems. For example, less frequent ‘mucking out’ could increase disease risk during housing (Results provide limited evidence that summer grazing is beneficial to cattle welfare. This is predominantly evidenced through behavioural indicators of welfare, with some support from physical indicators. Due to the behavioural data, this study was able to draw conclusions beyond those elsewhere, such as those reported by This study found evidence that summer grazing periods are beneficial to beef cattle welfare, compared to housing. This conclusion was predominantly borne out through behavioural assessments, highlighting not only the role grazing has in livestock production, but also how welfare must be approached holistically using multiple types of measures. Strong temporal differences in welfare, across numerous indicators, highlight the potential for negative welfare outcomes during winter housing and thus housing conditions are a key area for intervention to improve cattle welfare. The study also highlights the need to develop and standardise holistic approaches to animal welfare assessments that are inclusive of molecular techniques.Supplementary Material


Sequence constraints associated with topological energetics restrict the evolution and mutational tolerance of membrane proteins. Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain . These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains. Proteins exhibit considerable variation with respect to their tolerance of amino acid substitutions. This (in)tolerance constrains evolutionary trajectories and defines the extent to which proteins are sensitive to the effects of random genetic variation . The heat maps in Because of the intrinsic propensity of TM7 to adopt aberrant topologies . NeverthG protocol to estimate the energetic effects of all possible missense mutations on the stability of the native conformation (ΔΔG) based on the structure of rhodopsin  from their effects on the stability of the native rhodopsin fold (stage II folding). Therefore, to evaluate whether mutations in either TM domain are more likely to destabilize the native fold, we used the Rosetta ΔΔ similar . Slight thin TM2 . These dG predictor)  are similar in magnitude. However, because of the underlying difference in the hydrophobicity of these domains, the median transfer free energy among TM7 variants  is higher than that of the TM2 variants   , which scal/mol) . Given tn = 33), relative to those bearing charged residues in the N-terminal portion of TM7 . This observation provides additional evidence that the proteostatic effects of certain mutations within TM7 are likely to arise from their disruption of cotranslational folding transitions.Our previous findings on the cotranslational misfolding of TM7 provide an additional lens for the interpretation of mutagenic effects within TM7. We recently demonstrated that mutations within TM7 promote the formation of two competing non-native topomers during cotranslational folding, including one in which K296 snorkels into the membrane interface (near-native topomer) and one in which TM7 slips into the ER lumen  (Kd (dissociation constant) of 25 pM  bearing an N-terminal hemagglutinin (HA) tag (extracellular) followed by an internal ribosome entry site and a dasher GFP  on the following day (50 to 70% confluence) with a plasmid mixture containing the rhodopsin library and the Bxb1 recombinase expression vector . Transfections were typically carried out by mixing 7.6 μg of total plasmid with 29 μl of Fugene 6 in accordance with the manufacturer’s instructions. Transfected cells were then grown at 33°C for 4 days in full media. Doxycycline (2 μg/ml) was added to the media 1 day after transfection to induce expression of rhodopsin and eGFP. Stable cells expressing single rhodopsin variants were then isolated using a BD FACS Aria II  4 days after transfection based on their characteristic gain in bicistronic eGFP expression and coincident loss of BFP expression  from cell pellets using either a previously described protocol . We also removed intensity values derived from 50 or fewer reads across the four quartiles, as well as scores for any variants that were not consistently observed within each replicate of each experiment. The average number of reads associated with each variant across two biological replicates is shown in tables S3 and S4 for reference.To estimate surface immunostaining levels for each variant from sequencing data, we created a computational pipeline for the analysis of demultiplexed sequencing data. Briefly, next-generation sequencing data from each cellular isolate were first filtered to remove any reads in which the expected number of errors is ≥1  with an


Micro-doughLAB was used to study mixing and pasting properties. Doughs were characterised using small amplitude oscillatory measurements (SAOS), with a controlled stress rheometer, and regarding Texture Profile Analysis (TPA) by a texturometer. Dietary fibre content and its soluble and insoluble fractions were also evaluated on the developed breads. Acorn flour showed promising technological properties as food ingredient for gluten-free baking , being an important fibre source.Gluten is a fundamental ingredient in breadmaking, since is responsible for the viscoelastic behaviour of the dough. The lack of gluten has a critical effect on gluten-free dough, leading to less cohesive and less elastic doughs, and its replacement represents a challenge for bakery industry. However, dough rheology can be improved combining different ingredients with structural capacity and taking advantage from their interactions. Although acorn flour was used to bake bread even before Romans, nowadays is an underexploited resource. It presents good nutritional characteristics, particularly high fibre content and is naturally gluten free. The aim of this study was to use acorn flour as a gluten-free ingredient to improve dough rheology, following also market trends of sustainability and fibre-rich ingredients. Doughs were prepared with buckwheat and rice flours, potato starch and hydroxypropylmethylcellulose. Two levels of acorn flour (23% and 35%  Gluten is one of the most important ingredients in bread making. It is responsible for the viscoelastic behaviour of the dough . Gluten On the other hand, gluten-free dough (GFD) is unable to form a protein network similar to gluten. This raises several difficulties and thus the loss of quality on gluten-free bread baking . The lacRheology measurements are essential tools to understand the performance of the doughs, allowing to predict the final result and the before quality of bread . Among tNowadays, due to resource scarcity worldwide, is essential to use by-products and underexploited natural raw materials that are not valued enough in food production. This fact led to market trends of sustainability , naturalQuercus genus tree. These trees are the basis of the Sustainable Agriculture System named “Montado” in South of Europe. In Portugal, acorns are abundant and can be considered an underexploited resource, since nearly 50% of the fruits in the trees are not used or harvested [Quercus ilex and Quercus rotundifolia (holm oak). Since they are not so bitter comparing to other species, is not necessary to remove bitterness, as Korus et al. [Acorn is the fruit of arvested . Many auarvested ,26 statearvested  go furtharvested . This frs et al.  describes et al. , acorn fs et al. ,29.Comparing this work with the referred authors ,22 who hThe objective of this work was to study the impact of acorn flour from holm oak, on the rheology characteristics of the GF dough, and also on the dietary fibre content, both soluble and insoluble fractions, of the GF baked breads. Two levels of incorporation (23% and 35%) were considered.Quercus ilex and Quercus rotundifolia) , rice flour , potato starch , dried yeast , hydroxipropyl methylcellulose (HPMC) as a gelling agent , sugar, sunflower oil, salt, and water. The flour ingredients and the HPMC were kindly supplied for free, except yeast, sugar, oil and salt that were purchased from local market.Doughs formulations were prepared with the following flours: buckwheat flour , acorn flour from dried holm oak (w/w (sample A23%) and 35% w/w (sample A35%) of total flours mixture, respectively, 50% and 75% in relation to the buckwheat flour. The three formulations studied are summarised in Fernandes  developeMicro-doughLAB 2800  manufacturer’s protocol at 63 rpm speed  Method 54-21.01) was used to assess the optimum water absorption capacity for formulations with acorn flour. The peak value of torque of the optimised control-formulation was used as a reference (93 mN.m). Optimum water absorption values  were determined for the formulations containing acorn flour in order to achieve a peak of 93 mN.m ± 4%.The moisture of the different flours was measured through an automatic moisture analyser PMB 202 . The values were buckwheat flour—12.35%; acorn flour—8.03%; rice flour—11.08%; potato starch—18.10%.Ingredients were mixed in a Thermo processor equipment , initially to activate the yeast, by adding water, yeast and sugar for 2 min at 27 °C, at velocity 1. Following, the other ingredients were added and mixed for 10 min in a dough mixing program (wheat ear symbol). The resulting dough was divided in three portions and poured into three equal containers and placed in a fermentation chamber Arianna XLT133  for 50 min at 37 °C. All measurements were carried out after fermentation: three loaves of each formulation were prepared, and all the analyses were performed minimum in triplicate: three measurements were taken from independent three portions of each loaf. Mean values and standard deviations from each determination were recorded.In a parallel work, the impact of acorn flour addition in the bread characteristics was studied, using exactly the same dough formulations above described. After fermentation, the dough was baked in an electric oven Johnson A60  for 50 min at 180 °C. Correlations between dough properties and breadmaking characteristics (bread firmness) were analysed.The pasting properties of flour blends, according to each GFD formulation, were tested using a Micro-doughLAB 2800 , following the manufacturer’s cooking protocol with some modifications . ConstanFor dough characterisation, by dynamic rheometric measurements, formulations were prepared with all ingredients, as previously described. Rheological tests were performed using a controlled stress rheometer  coupled with a UTC-Peltier system. A serrated parallel-plate P20 (diameter of 20 mm) was used, with a gap between plates of 1 mm. Each sample was prepared in a small portion of dough, and after 50 min of fermentation at 37 °C in a fermentation chamber Arianna XLT133 , was allowed to rest for 30 min at 5 °C. The fermented dough samples were placed between the plates, and after its adjustment, the edges were covered with paraffin to protect the sample from dehydration during measurements. Rheometer temperature was kept at 5 °C during test performance to avoid dough fermentation. Samples were left for 15 min before the test, to allow residual stress to relax and to stabilise the temperature. All rheological measurements were run, at least, in triplicate.Small amplitude oscillatory shear (SAOS) tests were conducted to assess linear viscoelastic properties of the GFD. The dynamic linear viscoelastic region of each sample was previously determined through stress sweep tests at frequency of 6.28 rad/s (1 Hz). Frequency sweeps (from 0.0628 to 628 rad/s) were performed at constant shear stress of 10 Pa.Texture profile analysis (TPA) was performed after dough fermentation at 37 °C during 50 min, using a texturometer TA.XT.plus  equipped with a load cell of 5 kg and penetration mode of the equipment set up. Dough samples were placed in cylindric containers with 100 mm in diameter and a height of 80 mm. These cylinders were completely filled by the sample.The measurement conditions were 19 mm diameter acrylic cylindrical probe, 20 mm of penetration distance, 1 mm/s of crosshead speed and 5 s of waiting time between the two measurements cycles. Each measurement was repeated at least four times. Firmness and cohesiveness were the main representative texture parameters obtained from TPA, to characterise the dough, as it was reported by Nunes et al. .Dough colour was determined using a colorimeter Croma-Meter CR 400 , through the CIE L*a*b* system  using the following parameters: L*—lightness variable ; a*—intensity of green (−60 < a* < 0) or red (0 < a* < +60); and b*—intensity of blue (−60 < b* < 0) or yellow (0 < b* < +60). Each sample was measured in triplicate.pH measurements were performed in three different points of each fermentation dough, with a potentiometer pH-Meter Basic 20 , until stabilisation.According to Martins et al. , gluten-p < 0.05. Results are presented as mean values and standard deviations. Correlations were tested between the different variables, using the program Statistica 10.0 and the function Bivariate Scatterplot.Results were analysed using the statistical programme Origin Pro 8 . Experimental data were compared using analysis of variance (one-way ANOVA), and the Tukey test was used to evaluate mean differences at a confidence level of 95%, with significant differences considered to be Flour blends’ and pure flours’ pasting curves, obtained from the MidrodoughLab are represented in In Along the heating process, the starch granules retain water and swell, which results in viscosity increasing, until peak viscosity (C3) is reached and indicates water-binding capacity, and starch gelatinisation ,35. ContStarch gelatinisation depends on the starch characteristics and its swelling capacity. Moreover, the starches establish interactions with the HPMC, a fact that also contributes to the maximum viscosity of the dough, reached during gelatinisation . AccordiThe following stage is characterised by a viscosity decreasing to a minimum (breakdown). This happens when the granule absorbs as much water as to achieve its rupture point, while temperature keeps increasing . As we cWhen gelatinised starch temperature cools, viscosity increases until the end of the test (C5), the moment when we can see the formation of a gel, resulting from amylose retrogradation . RegardiQuercus rotundifolia and Quercus ilex from Cappai et al. [Since our study is focused on acorn flour, it is important to highlight that among the different species of oak trees, the acorn starch is also different due to taxonomic characteristics of the plant . For thii et al.  and Corri et al. .Finally, it is also important to mention the role that proteins have in the dough rheology behaviour, contributing as well to gluten replacing functions . DiffereThe mechanical spectra of the GF doughs  are presented in It is possible to observe in p < 0.05) for A23% dough was observed, meaning that the addition of acorn flour in dough formulation induced a significantly increase of elastic modulus, acting as a strengthening agent of the structure. However, the dough with 35% of acorn flour, reduces significantly G′ values, being similar to control dough. A similar effect will be found in the texture results, in terms of the TPA parameters. This fact indicates that the growing incorporation level of acorn flour presents a negative impact on the structure, since it is possible to observe a modification of the rheological behaviour, with the reduction of the elastic modulus at 62.8 rad/s. The aforementioned behaviour is not significantly different (p > 0.05) from the one of control dough, showing that the benefits, in terms of viscoelastic characteristics, induced from the addition of acorn flour are not extend to higher levels of incorporation. Regarding the improvement of rheology characteristics observed in acorn flour doughs, it is also important to underline the contribution of the fibre content present in acorn flour. Several authors have studied the impact of fibre in GFD formulation, with the objective of improving rheology. The dough formulation with the inclusion of fibre-rich ingredients, positively influenced rheological properties, with higher values of both moduli, and structuring the GF dough closer to a gel-like material [About G′ values at 62.8 rad/s, a significantly higher value  the firmness of the dough comparing with control. Between the two acorn flour doughs, the firmness is significantly (p < 0.05) higher in A23% when comparing with A35%. For the lowest level of incorporation, it was possible to obtain a more structured dough, already supported in terms of increasing the elastic modulus (G′), which corresponds to a higher firmness value. For the 35% of acorn flour incorporation, the extension of the network developed is weaker, resulting in a softer dough, similar to the control. Probably, the excess of acorn starch caused the weakening of this network, as we can observe from pasting curve analysis, where a noticeable decrease of acorn flour viscosity parameters is revealed.These findings are in accordance with the previous rheology measurements results discussion. Nevertheless, the firmness of the dough with 35% of acorn flour is significantly higher than the control dough. The results show that partial replacement of buckwheat flour by acorn flour improves the firmness of the dough, for either levels of acorn flour.p < 0.05) their cohesiveness, when compared with control dough. However, no significant differences were found between the two levels of acorn flour incorporation. From these results, it is possible to observe that the incorporation of acorn flour in the formulation, with the reduction of buckwheat flour share, increases the cohesiveness of the dough. Furthermore, Correia et al. [Cohesiveness is associated with the level of structure between the different elements that take part in the dough matrix . The obta et al.  also conThe increase of firmness and cohesiveness of the dough with acorn flour addition, together with the reduction of buckwheat flour incorporation level, can be explained due to the nutritional composition of acorn flour  in compaIn a parallel work, the impact of acorn flour addition in the bread texture was characterised, using exactly the same formulations and processing conditions used in the present study. In that work, it was found that acorn flour incorporation, generally, induces an increase in bread firmness. Through the results obtained in the present study, it was possible to establish a positive correlation between the bread firmness and the viscoelastic characteristics of the dough—G′ at 0.628 rad/s A and betThe obtained correlations, which allow to predict the behaviour of the bread, in terms of firmness, taking into account the dough rheological behaviour and its texture, are extremely useful in terms of the gluten-free bakery industry. Similar correlations have been obtained by other authors ,37.p > 0.05).p < 0.05) with the increase of acorn flour content. These results show a reduction in whiteness and an increase in the browning index, meaning the darkness of the dough. This effect on the dough is important, as it will contribute to the improvement of bread colour. This is a relevant parameter when observing consumers’ preferences. For GF bread, this characteristic is even more important, since it frequently presents pale colours when compared to their wheat counterparts, what is referred as a depreciative factor in GF bread [p < 0.05) increase with acorn flour incorporation, which means, respectively, a more intensive red than green, and a predominance of yellow over blue. Colour differences (ΔE) between control bread and acorn breads can be calculated by the following equation:Concerning dough colour, it is possible to observe that L* value has reduced significantly  the total fibre and insoluble fibre, comparing with control bread formulation. This was expected, since acorn is a good source of DF, particularly in the insoluble fraction [The results of GF bread fibre analysis, soluble fraction, insoluble fraction and total fibre are presented in fraction .p < 0.05), comparing with the control formulation, when acorn flour incorporation was considered. This fact can be explained since the soluble fibre fraction of acorn flour in comparison to buckwheat flour is much lower.However, it is important to note that the soluble fibre content of the breads significantly decreased (According to Melini and Melini , a healtFollowing USDA recommendations, the DF Reference Intake is 25 g/day for women and 38 g/day for men. If we consider an ingestion of 100 g of fresh bread per day, the 23% acorn flour bread would serve 7.5 g of fibre. This represents about 30% and 20% of daily adequate DF intake for women and men, respectively, meaning that acorn flour bread can be an important vehicle of fibre, with the possibility to use the respective nutritional claim.Thus, acorn flour seems to be a very promising ingredient, in order to improve DF content in GF bread.Based on the obtained results, it can be stated that acorn flour significantly affects the rheology properties of the doughs. Moreover, acorn flour has an impact on the dough’s mixing and pasting curves and improved the firmness, the cohesiveness and the viscoelasticity of the fermented dough. The 23% of acorn flour incorporation presented better results when comparing with 35%. According to SAOS, GF doughs exhibit a weak gel-elastic like behaviour with G’ values higher than G’’ and frequency dependent. Acorn flour incorporation caused the acidification and increased the darkness of the dough, that will have a positive impact in terms of sensory appreciation of the bread. Thus, acorn flour can be a very promising ingredient, in order to improve both rheological GF dough properties and nutritional GF bread quality, in particular DF content, a really important nutrient in special requirement diets.


Whether creativity is a domain‐general or domain‐specific ability has been a topic of intense speculation. Although previous studies have examined domain‐specific mechanisms of creative performance, little is known about commonalities and distinctions in neural correlates across different domains. We applied activation likelihood estimation (ALE) meta‐analysis to identify the brain activation of domain‐mechanisms by synthesizing functional neuroimaging studies across three forms of artistic creativity: music improvisation, drawing, and literary creativity. ALE meta‐analysis yielded a domain‐general pattern across three artistic forms, with overlapping clusters in the presupplementary motor area (pre‐SMA), left dorsolateral prefrontal cortex, and right inferior frontal gyrus (IFG). Regarding domain‐specificity, musical creativity was associated with recruitment of the SMA‐proper, bilateral IFG, left precentral gyrus, and left middle frontal gyrus (MFG) compared to the other two artistic forms; drawing creativity recruited the left fusiform gyrus, left precuneus, right parahippocampal gyrus, and right MFG compared to musical creativity; and literary creativity recruited the left angular gyrus and right lingual gyrus compared to musical creativity. Contrasting drawing and literary creativity revealed no significant differences in neural activation, suggesting that these domains may rely on a common neurocognitive system. Overall, these findings reveal a central, domain‐general system for artistic creativity, but with each domain relying to some degree on domain‐specific neural circuits. Despite some progress, however, the neural mechanisms of creativity remain incompletely defined. One reason relates to variability in how creativity is defined, leading to heterogeneity of measurement and evaluation across multiple domains of performance. Previous studies suggest that a wide range of brain regions are recruited during different creative thinking tasks, such as divergent thinking, artistic creativity, and insight, raising questions about the existence of a domain‐general neurocognitive system for creative thinking, beyond diffuse prefrontal activation  that depend upon visual mental imagery , including hippocampus, parahippocampus, and fusiform gyrus, exhibits greater recruitment during drawing creativity, such as visual art design  meta‐analyses on three artistic creative modalities: music, drawing, and literary. First, we applied ALE meta‐analyses to identify brain activation patterns for each creative modality. Then, we conducted conjunction and contrast analyses of these meta‐analytic maps to assess the common and distinct neural correlates supporting the three artistic forms. In terms of previous fMRI meta‐analyses on verbal and visuospatial creative thinking, as well as relevant reviews  studies on artistic creativity up to July 14th, 2019. Three artistic modalities involving creativity were included in the meta‐analysis: musical creativity, drawing creativity, and literary creativity. For each modality, all relevant search terms were combined (“AND”) with “fMRI” or “PET.” Specifically, the keywords for musical creativity included “musical improvisation,” “musical creativity,” “music creativity,” and “compose AND creativity.” This search yielded a total of 140 studies; after removing duplicates, 110 studies were retained. For drawing creativity, the keywords included “drawing creativity,” “visual–spatial creativity,” “visual creativity,” “visual divergent thinking,” and “figural creativity.” In addition, one fMRI study published using Chinese‐language was included. This search yielded a total of 128 studies; after removing duplicates, 93 studies were retained. For literary creativity, the keywords included “creative writing,” “poetry,” “story and creativity,” “writing and divergent thinking,” and “metaphor creativity.” This search yielded a total of 103 studies; after removing duplicates, 73 studies were retained.Further inclusion criteria for these candidate studies were the following  across 15 studies; drawing creativity studies included 210 foci from 13 experiments with 252 participants  across 10 studies; and literary creativity included 169 foci from 14 experiments with 199 participants  across seven studies. More details of the included literature can be seen in Table 2.2http://brainmap.org), which is a freely available and quantitative meta‐analysis method  with a cluster forming threshold of p < .001 , we also performed conjunction analyses and contrast analyses in GingerALE. Due to the exploratory nature of the analysis, the correction for pairwise contrast analyses was loosely defined at an uncorrected 2.3ric.uthscsa.edu/mango) and MRIcron software (http://www.sph.sc.edu/comd/rorden/mricron).All significant clusters were reported, including the volume, coordinates in MNI space, and Z‐scores at peaks. For visualization purposes, these results were registered onto an MNI‐space template  brain using Mango , and they required performance on MR‐compatible instruments during functional imaging. A meta‐analysis of 21 musical creativity experiments showed a subset of activated clusters associated with musical improvisation , IPL, and superior occipital gyrus (SOG); two clusters were also found in the right hemisphere: MFG and IFG , IFG, lingual gyrus, parahippocampal gyrus, and medial dorsal nucleus; two clusters were also found in the right hemisphere: IFG and cerebellum posterior lobe , left DLPFC , and right IFG , right DLPFC, and right IFG. No significant activation differences were found between drawing creativity and literary creativity . Second, we aimed to identify distinct regions with respect to each creative domain. Overall, the results of the meta‐analyses revealed three prefrontal brain regions common to music, drawing, and literary creativity: left pre‐SMA, left DLPFC, and right IFG. Moreover, contrasting these modalities revealed reliable domain‐specific activation for each artistic domain: music creativity (improvisation) recruited bilateral IFG, left PreG, and left MFG compared to the other two artistic modalities; drawing creativity recruited the right MFG, left fusiform gyrus, left precuneus, and right parahippocampal gyrus compared to musical creativity; and literary creativity recruited the left angular and right lingual gyrus compared to musical creativity. Together, these meta‐analytic findings indicate that a set of prefrontal brain regions support creative performance across diverse artistic domains, but that specific regions also support creativity in each creative domain. In the following sections, we discuss the potential functional significance of these converging findings for understanding artistic creativity, focusing on current theories of creativity as a domain‐general process.4.14.1.1y = 0) crossing the anterior commissure  across three artistic domains belonged to the pre‐SMA. We thus propose that pre‐SMA may also be involved in higher‐order cognitive processes during artistic creativity involved in planning and selecting complex action sequences  has consistently implicated the ventrolateral PFC , fundamental cognitive mechanisms , and the interplay between these cognitive processes (Mekern, Hommel, & Sjoerds, 5Whether creativity relies on domain‐general or domain‐specific cognitive processes remains an open question. Many behavioral studies have explored potential domain‐general and domain‐specific mechanisms by investigating associations between distinct creative tasks. Recent neuroimaging research has demonstrated consistent prefrontal activation during verbal, visuospatial, and musical creativity, but a systematic framework to interpret these findings has so far been lacking. Such a neuroscience framework can enrich current theories of creativity and motivate a promising direction to contextualize future work on the neurocognitive basis of creative thinking. The present study aimed to uncover the domain‐mechanisms of artistic creativity by performing an ALE meta‐analysis on brain regions associated with three forms of creative performance. Consistent with our hypotheses, results revealed that the three creative domains all recruited the pre‐SMA, left DLPFC, and right IFG, suggesting that these regions support domain‐general processes during artistic creation. We also found that some regions were more specialized for one type of artistic creativity, such as MTG and right lingual gyrus for literary creativity, suggesting that domain‐specific processes are also important for artistic creativity. Taken together, these findings provide a path forward for future investigations of artistic creativity, emphasizing the need to dissociate domain‐general vs. domain‐specific neural systems underlying creative performance.The authors declare that they have no conflict of interest.


J-domain proteins (JDPs), obligatory Hsp70 cochaperones, play critical roles in protein homeostasis. They promote key allosteric transitions that stabilize Hsp70 interaction with substrate polypeptides upon hydrolysis of its bound ATP. Although a recent crystal structure revealed the physical mode of interaction between a J-domain and an Hsp70, the structural and dynamic consequences of J-domain action once bound and how Hsp70s discriminate among its multiple JDP partners remain enigmatic. We combined free energy simulations, biochemical assays and evolutionary analyses to address these issues. Our results indicate that the invariant aspartate of the J-domain perturbs a conserved intramolecular Hsp70 network of contacts that crosses domains. This perturbation leads to destabilization of the domain-domain interface—thereby promoting the allosteric transition that triggers ATP hydrolysis. While this mechanistic step is driven by conserved residues, evolutionarily variable residues are key to initial JDP/Hsp70 recognition—via electrostatic interactions between oppositely charged surfaces. We speculate that these variable residues allow an Hsp70 to discriminate amongst JDP partners, as many of them have coevolved. Together, our data points to a two-step mode of J-domain action, a recognition stage followed by a mechanistic stage. It is well appreciated that Hsp70-based systems are the most versatile among molecular chaperones—functioning in all cell types and in all subcellular compartments. Via cyclic binding to protein substrates, Hsp70s facilitate their folding, trafficking, degradation and ability to interact with other proteins. Hsp70 function, however, depends on transient interaction with J-domain protein cochaperones that not only deliver substrates, but also activate the structural changes needed for efficient Hsp70 binding to substrate. But how J-domain proteins mechanistically function to drive these changes and how an Hsp70 discriminates among multiple J-domain partners have remained challenging central questions. Here, by using a combination of computational, evolutionary and experimental approaches, we provide evidence for a two-step mechanism. The initial recognition step involves variable residues that allow fine tuning of both the specificity and strength of J-domain protein interaction with Hsp70. The second, that is the mechanistic step, involves conserved residues that act to disrupt a conserved network of intramolecular interactions within Hsp70, thus ensuring robust activation of the structural changes necessary for effective substrate binding. We suggest that our findings are likely applicable to most Hsp70 systems. By transiently binding many different polypeptide substrates, Hsp70 chaperones assist in diverse cellular processes, from de novo protein folding to protein trafficking to disassembly of protein complexes. J-domain protein (JDP) cochaperones are crucial players in the cycle of interaction of Hsp70 with substrate. They transiently interact with ATP-bound Hsp70 via their defining J-domain. This interaction, in coordination with substrate binding, facilitates stimulation of Hsp70's ATPase activity, which in turn drives the large scale conformational changes that stabilize substrate interaction –4.The structure of both J-domains and Hsp70s are conserved. J-domains are composed of four α-helices. Helices II and III form a finger like structure. The helix II/III connecting loop includes a conserved histidine, proline, aspartate tripeptide (HPD). These invariant residues are critical for J-domain stimulation of Hsp70's ATPase activity. Hsp70s are more complex in structure, containing two large domains—a nucleotide binding domain (NBD) and a substrate binding domain (SBD)—connected by a “linker” segment. When ATP is bound, the domains are docked such that the substrate binding site in the β subdomain of the SBD (SBDβ) is easily accessible , 6. UponThe heart of the allosteric control mechanism behind these Hsp70 conformational transitions is an interaction network at the interdomain interface –12. A crEscherichia coli JDP DnaJ and Hsp70 DnaK established an arginine at the Hsp70 NBD/SBDβ,linker interface, R167 of the NBD, as an important for J-domain function [It has been clear for years that J-domain interaction is essential for stimulation of Hsp70’s ATPase activity . Particufunction , 20, 21.function . This nofunction , the J-dE. coli and the mitochondrial Hsc20/Ssq1 of Saccharomyces cerevisiae [Despite the accumulated information about J-domain/Hsp70 interactions, important questions remain. Although the end result of ATP hydrolysis is destabilization of Hsp70 domain-domain interactions, the initial structural consequences of J-domain binding are not known. Furthermore, there is no understanding of what determines the specificity of J-domain/Hsp70 interactions and how Hsp70s discriminate amongst their multiple JDP partners. To address these issues, we investigated two JDP/Hsp70 pairs: DnaJ/DnaK of revisiae , 25, whiResults of our analyses point to two previously unappreciated facets of J-domain/Hsp70 interactions, leading us to propose a two-step model of J-domain action–recognition, followed by mechanistic action. Molecular simulations point to an active role of the invariant aspartate of the J-domain HPD in the mechanistic step–perturbing the intramolecular interaction network formed by the previously identified, key NBD arginine. This perturbation promotes allostery-related conformational changes of Hsp70. Evolutionary analyses indicate that J-domain/Hsp70 recognition, is dependent on variable, coevolving residues, even though the general mode of interaction of J-domains with Hsp70s is the same. This variability provides a plausible explanation for how Hsp70s are able to discriminate amongst their multiple JDP partners, thus enabling evolution of complex JDP/Hsp70 interaction networks.JD-DnaK) [NBD throughout), the arginine that previous analyses of DnaK established as important for allosteric transitions [To obtain a structural model of the JDP-Hsp70 complex formed by Hsc20 and Ssq1, we combined protein-protein docking with all-atom molecular dynamics (MD) simulations see , S2 Fig.JD-DnaK) . Helicesnsitions , 20, 21.NBD because of its demonstrated importance in NBD/SBD communication [NBD to be capable of forming two ion pairs, one with D517 of the SBDβ (referred to as DSBD throughout) and one with D429 of the interdomain linker (referred to as DLk throughout). To analyze these two contacts of RNBD more closely, we constructed the free energy map governing their formation  for interaction with DHPD, rather than simultaneous interaction with the aspartates on the SBD and the linker, DSBD and DLk. The observed differences in the depth of the free energy minima between Ssq1 and DnaK could be attributed to differences in the amino acid sequences or to the fact that Hsc20 was modeled as a whole instead of only its J-domain. Finally, an example of spontaneous transition of RNBD to the intermolecular contact with DHPD, captured in our unbiased equilibrium MD simulation of the Hsc20-Ssq1 complex, is shown in HPD perturbs the RNBD -centered interaction network at the NBD/SBDβ,linker interface.To assess the effect of J-domain binding to Hsp70 on this conserved arginine-centered interaction network, we carried out the same set of calculations for Ssq1 and DnaK in complex with Hsc20 and DnaJectively . For botJ-domain ; beside ith DHPD . In thes affects the compactness of the NBD/SBDβ,linker interface. Umbrella sampling was used because it allows testing of conformations not accessible by conventional MD simulations. For these simulations a center of mass distance between SBDβ and its NBD interacting region (SBDβ-NBD distance) was used as a relevant coordinate. To accelerate the free energy convergence and to allow exhaustive sampling of interdomain contacts these simulations were performed using Ssq1 and DnaK with a truncated SBDα subdomains.Because of these effects on interdomain communication, we used MD simulations to probe how weakening of the cross-domain interactions of RNBD->D variants , both DnaK and Ssq1 adopt conformations with markedly reduced number of NBD/SBDβ contacts. In particular, upon J-domain binding, the populations of DnaK and Ssq1 that retain at least 70% of interdomain contacts are reduced from 88% to 50% and from 91% to 45%, respectively. A similar reduction is observed for the RNBD->D substitution in the absence of J-domain, indicating that perturbation of the arginine-centered interaction network promotes partial disengagement of SBDβ. These results also indicate that J-domain binding perturbs the RNBD intramolecular interactions, which in turn leads to partial disengagement of SBDβ, while the interdomain linker remains tightly associated with the NBD. Such an arrangement favors ATP hydrolysis, which triggers further conformational changes [To assess what structural changes took place within the NBD/SBDβ, linker interface we used umbrella sampling trajectories to determine the number of interdomain contacts . We anal changes , 14, 15.NBD-centered interaction network is the same in DnaK and Ssq1, in that RNBD interacts with homologous aspartates in the linker and SBDβ. To investigate the prevalence of these residues, we analyzed Hsp70 sequences from a large number of bacterial and eukaryotic species. RNBD and its interaction partner DLk is invariant in our data set  residue-residue contacts, and computed the relative energetic contributions of individual residues to complex stability . The predominant contributors are eight charged residues  that form a network of electrostatic interactions with four negatively charged residues of the Ssq1 NBD  and K70JD of helix III that promotes complex formation, though its binding partner(s) was not identified in our analyses.Having found similarities of the Rensively ; both potability , S8 Fig.residues . Three pWe carried out two types of biochemical experiments to verify the functional importance of key residues that based on our computational analysis form the Hsc20/Ssq1 binding interface. First, we asked if alanine substitution of these Ssq1 residues disturbs the Hsc20/Ssq1 interaction. To enable detection of the inherently transient J-domain/Hsp70 interaction, we used Hsc20 having a site-specific p-benzoyl-l-phenylalanine (Bpa) crosslinker  replacinThe functional importance of charged residues in Hsc20/Ssq1 interaction led us to consider whether the initial encounter of the two proteins is electrostatically driven—an idea furthered by the presence of a patch of uniformly positive electrostatic potential around helix II of the J-domain and a complementary patch of negative potential around subdomain IIa of the Ssq1 NBD, the site of J-domain interaction . MD simuTo examine the driving forces behind spontaneous Hsc20/Ssq1 interaction, we computed the underlying free energy profile using umbrella sampling. The resulting profile , the freJD/DnaK interface, and of these 12, only 3 are occupied by the same amino acids in the two interfaces. A broader comparison of Hsc20, Ssq1, DnaJ, and DnaK with their orthologs from a wide range of species belonging to fungi, animals and plants  and SBDβ subdomain (DSBD) of Hsp70. This displacement promotes destabilization of the interdomain interface, such that the SBDβ partially disengages from the NBD. These results are consistent with experimental data showing less protection against hydrogen exchange of this region when residues in the NBD/SBDβ interface are mutated [NBD centered interaction network. These perturbations could also modify the interaction mode of ATP bound to Hsp70, thus affecting its rate of hydrolysis—a hypothesis that requires further studies to verify. Overall, we postulate that the substrate and J-domain dependent allosteric pathways converge and that this convergence allows synchronization—only upon simultaneous interaction of substrate and J-domain is ATPase activity effectively stimulated, driving transition to the undocked state and substrate capture.Based on detection of an energetically favorable interaction between invariant D mutated  It is im mutated  because  mutated , 34, 35. mutated , 11, 36.SBD replaced by asparagine, pointing to the importance of the RNBD centered interactions for the allostery of Hsp70s.This coordinated mechanism also impacts the larger picture of Hsp70 allosteric dynamics. All Hsp70s studied share major characteristic transient conformational shifts–for example, undergoing repeated transitions involving undocking from the NBD of both SBDβ and SBDα. However, recent single molecule experiments indicate that the frequency of such transitions differs among members of the Hsp70 family , 37–39. HPD- RNBD interaction per se has been previously put forth, the functional connection between these residues is well supported by genetic and biochemical data. Alteration of either RNBD or residues interacting with it (DSBD and DLk) [NBD(R167H) of DnaK suppresses nonfunctional DHPD(D35N) variant of DnaJ [HPD is the only mechanistic effect of J-domain binding to Hsp70. For example, the invariant histidine and/or proline of the HPD may play specific roles, and, as previously suggested [NBD is key and could be considered as an allosteric perturbation site for the J-domain action.Though no model for the mechanistic role of the Dand DLk) , 36, 40 and DLk)  and Hsc2 of DnaJ . Howeveruggested , conformAt first glance, the sequence diversity of the interface of J-domain-Hsp70 complexes, but maintenance of overall positioning, and thus, function, seems surprising. It raises the question of how this diversity evolved without loss of function. Our analyses suggest that functional J-domain/Hsp70 interactions have been maintained over time via coevolution of residues that form the binding interface. The nature of the Hsc20/Ssq1 interface is instructive in this regard. Each key residue on one side of the large interface contacts more than one residue on the opposite side. Such a multifaceted interaction network is highly flexible. Over time residues can be added to or deleted from the interface without ever losing the ability to maintain functional interaction between the coevolving partners, enabling modulation of both the specificity and the strength of the interaction. Coevolution could also explain how a JDP might have been able to switch Hsp70 partnership during evolution, such as occurred in yeast mitochondria—switching of Hsc20 from interacting with a general multifunctional Hsp70 (Ssc1) to a specialized Hsp70 (Ssq1) . A JDP tWhat effect might this diversity have had on the functionality of Hsp70 chaperone systems more generally? We suggest that it has allowed evolution of complex Hsp70/JDP networks, both in situations in which a single type of Hsp70 interacts with many different JDPs and in which more than one Hsp70 coexist in a cellular compartment. In the first situation, replacement could well modulate the strength of a JDP/Hsp70 interaction, explaining why some JDPs have "easier access" to their partner Hsp70 than others. Here, electrostatic interactions could play a particularly important role. A JDP with a highly charged J-domain binding face would be able to recognize its Hsp70 partner at a longer distance, giving it priority over others in forming a productive interaction. In the second situation, the diversity serves to "insulate" Hsp70 networks, each Hsp70 interacting effectively only with its own subset of JDPs. For example, it could explain how yeast cytosolic Hsp70s Ssa and Ssb, which share common ancestry, evolved into independent networks, each with its own JDP partners , 45.JD/DnaK interface being a mosaic of electrostatic and hydrophobic interactions [In the two examples studied here, the predominant J-domain interactions are via helix II. However, their binding interfaces differ—the specialized Hsc20/Ssq1 interface involving mostly electrostatic interactions and the DnaJractions , 41, 46.ractions , 47 and ractions . As onlyOverall our data suggests a two-step process of J-domain function: initial binding that forms similar poses via highly variable residue-residue contacts, followed by a mechanistic step in which the invariant HPD perturbs the Hsp70 NBD-SBDβ,linker interface to foster a key allosteric transition. This separation could have future importance beyond a basic understanding of molecular chaperone function. It has long been appreciated that Hsp70 systems impinge on many biological functions and thus, not surprisingly, connected to many pathologic conditions raging from cancer, metabolic diseases to aging , 50. As + and Cl- ions were adjusted to 0.15 M. Temperature was kept at 310 K with the v-rescale algorithm [All simulations were performed using Gromacs 5  with thelgorithm  using a lgorithm  with a clgorithm  was usedSsq1 model was built with I-TASSER  using thThe structures of Hsc20 (PDB ID 3UO3) and Ssq1 (homology model) used for molecular docking were first subjected to unbiased MD simulations to sample global protein structural dynamics. The Hsc20 simulation system was composed of a single protein in a 9.2 nm × 9.2 nm × 9.2 nm dodecahedron box filled with ~17000 water molecules, while the Ssq1 simulation system was composed of a single ATP-bound protein solvated with ~48,500 water molecules in a 13 nm × 13 nm × 13 nm dodecahedron box. Both systems were simulated using CHARMM36  for 10 a2+ ion in Mg-ATP were kept. Both systems were simulated for 1 μs and the final structures from these simulations were used as initial configurations for the subsequent free energy computations.The systems were composed of either DnaK (PDB ID 4B9Q) or DnaK in complex with the J-domain of DnaJ (PDB ID 5NRO) placed in 13 nm × 13 nm × 13 nm dodecahedron box and solvated with approx. 52,000 water molecules. Crystal water molecules coordinated to MgNBD (R207 in Ssq1 and R167 in DnaK) were computed with well-tempered multiple walkers 2D-metadynamics [NBD interactions, the simulation systems contained either Hsc20-Ssq1 or DnaJJD-DnaK complexes or individual proteins, Ssq1 or DnaK, respectively, placed in a 11.8 nm × 11.8 nm × 11.8 nm dodecahedron box filled with ~34,000 water molecules. To make the system significantly smaller and thus achieve better convergence of the free energy maps, the lid sub-domain, SBDα, was truncated by removing residues 570–657 of Ssq1 and 535–602 of DnaK. As two coordinates for 2D-metadynamics, we used the center of mass (COM) distances between the guanidine group of the RNBD residue and the carboxylic groups of the partner aspartate residues at the SBDβ (D517 in Ssq1 or D481 in DnaK) and at the interdomain linker (D429 in Ssq1 or D481 in DnaK). Biasing potential was added every 10 ps with the bias factor of 15, a height of 0.1 kJ and a width of 0.04 nm. The sampled range of both coordinates was restricted to a 0.3–1.9 nm interval by applying one-sided harmonic potentials with a force constant of 3000 kJ nm-2. For both systems, 10 independent 1-μs-long walker-simulations were carried out. The final free energy maps were averaged over 15 individual maps computed in 25-ns intervals from the last 350 ns of the simulations to average out free energy fluctuations [The free energy landscapes describing the formation of intramolecular ion pairs by the critical Rdynamics . In ordetuations .NBD) guanidine group and the carboxylic groups of the aspartate residues in the linker (DLk) and in the SBDβ (DSBD) and ii) the COM distance between the RNBD guanidine group and the carboxylic group of the DHPD  and the linker  to an intermolecular contact with the aspartate in the HPD  were computed using well-tempered multiple walkers metadynamics. The sampled coordinate was defined as the difference between the two distances: i) the COM distance between the arginine (RDHPD see . To speeJD-DnaK) and free proteins (Ssq1 and DnaK) were performed in a 11.8 nm × 11.8 nm × 11.8 nm dodecahedron box filled with ~36,000 water molecules. The reaction coordinate was defined as the COM distance between the Cα atoms of SBDβ (residues 434–541 in Ssq1 and 395–505 in DnaK) and the Cα atoms of the NBD region at the interface with SBDβ . To accelerate the free energy convergence, the lid sub-domain (SBDα) was truncated (residues 542–657 in Ssq1 and 506–602 in DnaK). For all systems, the initial configurations for REUS simulations were alternately selected from two independent 200 ns steered-MD simulations. In these simulations, the distance between SBDβ and NBD was gradually increased using a moving one-sided harmonic potential with a force constant of 2500 kJ nm-2, applied to either the COM distance or the minimal distance between the Cα atoms of SBDβ and the NBD interfacial residues. The reaction coordinate in the 2.5–2.9 nm range was divided into 5 equally spaced windows in which the system was restrained using a harmonic potential with a spring constant of 2500 kJ nm-2. The same approach was used to study the effect of the RNBD->D substitutions (Ssq1(R207D) and DnaK(R167D)) on the relative arrangement of NBD and SBDβ. To perform these simulations, the RNBD residue was replaced by aspartate in all initial structures. For each of the REUS windows at least 800 ns long simulation were performed. Exchanges between neighboring umbrella sampling windows were attempted every 2 ps. The first 100 ns of the trajectory was discarded and free energy profiles were computed using WHAM [iU(r)−iF)/kT), where iU(r) denotes the value of the biasing potential for a given frame and iF denotes the free energy added to the i-th US window by the applied bias.The free energy profiles describing the compactness of the NBD, linker and SBDβ interface in Ssq1 and DnaK in the presence and absence of J-domain were computed with replica-exchange umbrella sampling (REUS). The simulations of the protein complexes  Ion pairs between Hsc20 and Ssq1 were identified using a cut-off of 0.5 nm for the distance between the centers of mass of the interacting charged groups . (ii) HyTo study spontaneous binding between Hsc20 and Ssq1, the simulation dodecahedron box of size 16.2 nm × 16.2 nm × 16.2 nm containing single copies of Hsc20 and ATP-bound Ssq1 solvated with approx. 96500 water molecules was prepared. The proteins were initially placed such that the COM distance between positively charged residues of the Hsc20 J-domain helix II  and negatively charged residues of Ssq1 NBD  was equal to 3 nm. For this system, five independent unbiased MD simulations were performed which were terminated after 60 ns as this time was sufficient for the native complex to spontaneously form in 2 out of 5 replicas. The free energy profiles for Ssq1 interaction with Hsc20 and with Hsc20 substitution variant were computed using replica-exchange umbrella sampling (REUS) . As a reProtein sequences were retrieved from OMA orthology database : Hsc20 . To establish the threshold for the ΔAIC value to be accepted as evidence for coevolution [thpercentile of this expected ΔAIC distribution provided thresholds to consider the observed ΔAIC for Coev model to be accepted as coevolving with p<0.01 confidence level.Maximum likelihood implementation of the Coev model  was usedvolution , we simuvolution  for thisE. coli strain C41(DE3) carrying two plasmids: pET21d encoding Hsc20 Bpa variant and pSUPT BpF encoding an engineered tRNA synthetase and tRNACUA for p-benzoyl-phenylalanine (Bpa) incorporation. Cells were cultured in the M9 minimal medium. Protein expression was induced by adding isopropyl-1-thio-D-galactopyranoside (IPTG) at a final concentration of 0.2 mM, L-arabinose at a final concentration of 0.2% (v/v) and Bpa at a final concentration of 0.1 mM. Cells were harvested and lysed using a French press in buffer J1 , 10% glycerol, 30 mM imidazole, pH 8.0). After a clarifying spin, the supernatant was precipitated with ammonium sulfate (0.35 mg/ml). After centrifugation, the pellet was resuspended in buffer J1 and dialyzed overnight against buffer J1. Next, the proteins were subjected to HisBind Resin (Novagen) chromatography. After sequential washing steps with buffers J1, J2  and J1, proteins were eluted with a linear 30–300 mM imidazole gradient in buffer J1. Fractions containing Hsc20 were collected, pooled and dialyzed against buffer J3  glycerol; 75 mM NaCl; 0.05% Triton X-100; 5 mM β-mercaptoethanol). After dialysis proteins were loaded on a P11 cellulose column (Spark Scientific Ltd), washed with a 150 mM NaCl solution in J3 buffer and eluted with a linear 150–800 mM NaCl gradient in J3 buffer. Fractions containing the purest Hsc20 were collected, pooled and dialyzed against the final buffer . Aliquots were stored at –70°C.Expression of Bpa-containing Hsc20 variants (Q46Bpa and M51Bpa) with a polyhistidine tag at the C-terminus was performed using the E. coli strain C41(DE3) was used for expression. Ssq1 variants were purified as described [Chaetomium thermophilum (Isu1Ct) was purified as described [All other Hsc20 variants with a polyhistidine tag at the C-terminus were purified according to , except escribed . Mge1 waescribed . Isu1 frescribed In all cases, protein concentrations, determined by using the Bradford (Bio-Rad) assay system with bovine serum albumin as a standard, are expressed as the concentration of monomers.PVKLHC) derived from the Isu1 sequence containing the PVK motif that interacts with substrate binding site of Ssq1 [2, and 10% (v/v) glycerol). Reactions (15 μl) were initiated by the addition of ATP  to a final concentration of 120 μM. Incubation was carried out at 25°C, and the reaction was terminated after 15 min by the addition of 100 μl of 1M perchloric acid and 1mM sodium phosphate. After addition of 400 μl of 20 mM ammonium molybdate and 400 μl of isopropyl acetate, samples were mixed and the phases were separated by a short centrifugation. An aliquot of the organic phase (150 μl), containing the radioactive orthophosphate-molybdate complex, was removed and radioactivity was determined by liquid scintillation counting. Control reactions lacking protein were included in all experiments. Values were plotted in GraphPadPrism. The ATPase activity corresponding to the maximal stimulation (Vm) of the wild-type Hsc20 was set to 1.The ATPase activity of Ssq1 variants defective in the Hsc20/Ssq1 interaction was measured as described by  with 0.5 of Ssq1 , 0.5 μM Ct at 3 μM and Hsc20 at 3 μM. Reactions were initiated by mixing ATP with proteins and then incubating at 23°C. NADH absorbance was measured at 340 nm for 500 seconds in JASCO V-660 spectrophotometer.The ATPase activity of the Ssq1(R207D) variant was measured using an enzyme-coupled spectrophotometric assay . Reactio2, and 10% (v/v) glycerol). Reactions were initiated by the addition of ATP to a final concentration of 2 mM and incubated at 25°C for 15 min. Reactions were then exposed to 254 nm UV light (CL-1000 Ultraviolet Crosslinker) for 7 minutes. Control reactions were not exposed to UV light. Next, 5 μl of 4-fold concentrated Laemmli sample buffer  glycerol) was added to the reaction mixes. After 10 min incubation at 100°C, the total reaction volume was loaded onto SDS-PAGE and visualized by Coomassie blue stain. Every experiment was replicated three times.Reaction mixtures (20 μl) containing 20 μM Hsc20 Bpa-containing variants and 5 μM Ssq1 variants were prepared in the reaction buffer  and proteins were digested overnight with 10 ng/μl trypsin  in 25 mM NH4HCO3 [Gel pieces were dried with acetonitrile and subjected to reduction with 10 mM dithiothreitol in 100mM NH NH4HCO3 , with thS1 FigRepresentative structures of Ssq1 (a) and Hsc20 (b) from our unbiased molecular dynamics simulations carried out using the CHARMM36 (left) and AMBER99SB-ILDN (right) force fields. Ssq1 and Hsc20 were simulated for 2 and 10 μs, respectively, with CHARMM, and for 2 and 5 μs, respectively, with AMBER. The obtained trajectories were subject to cluster analysis with an RMSD cutoff of 0.7 nm (Ssq1) and 0.35 nm (Hsc20). The centroids of each cluster were superimposed and are shown in different colors. NBD, SBDa and SBDb domains (in Ssq1) and J-domain and Isu1 binding domain (in Hsc20) are indicated.(PDF)Click here for additional data file.S2 Fig(a) Five most populated binding poses of Hsc20 along with their percentage contributions to the bound- state ensemble of the Hsc20-Ssq1 complex, obtained by the combined docking/MD simulation approach. Helices II and III are shown in cyan and the rest of Hsc20 in transparent cyan. SBD is in light brown, Ia and IIa subdomains of NBD are in light and dark grey, respectively, linker in yellow. (b) Time evolution of the average RMSD of helices II and III of the J-domain with respect to the center of the most populated binding pose above (the dominant bound state). Averaging was done over all 18 independent MD trajectories that were found to visit the dominant bound state at least once over the course of 500 ns. The shaded area shows the standard deviation of the RMSD.(PDF)Click here for additional data file.S3 FigContact maps of Ssq1 R207 and DnaK R167 calculated from 2D metadynamics trajectories in absence (grey bars) and presence (cyan bars) of Hsc20 and DnaJ J-domain, respectively. The contact is defined with a cutoff distance of 0.5 nm between non-hydrogen atoms, and the contacts with probability above 20% in either state are shown.(PDF)Click here for additional data file.S4 FigSteady-state ATPase activity of Ssq1 was measured alone, or in the presence of indicated proteins, using an enzymatically coupled assay. Bars represent average value for three independent measurements with error bars as standard deviation. Concentration of components: 1 μM Ssq1; 3 μM Hsc20; 3 μM Isu1; 1 μM Mge1; 1 mM ATP.(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigScale is in amino acid substitutions per position.(PDF)Click here for additional data file.S7 Fig(a) Probabilities (p) of the most stable ion pairs across the J-domain/Ssq1 interface, calculated from the 10.5 μs trajectory of the dominant bound state. (b) Distance distributions of the ion pairs shown in (a).(PDF)Click here for additional data file.S8 Fig(a) Probabilities (p) of the most stable hydrogen bonds across the J-domain/Ssq1 interface, calculated from the 10.5 μs trajectory of the dominant bound state. (b) Relative energy inputs to Hsc20-Ssq1 complex binding energy calculated with MM/PBSA for the residues of the Hsc20 J-domain (top) and Ssq1 (bottom).(PDF)Click here for additional data file.S9 Fig(left) Purified Hsc20 Q46Bpa and M51Bpa variants, were incubated with Ssq1 WT in the presence of ATP, irradiated with UV light and separated by SDS-PAGE. Indicated bands (1–8) were excised and subjected to trypsin digestion followed by LC-MS. Migrations of size standard, in kDa, are indicated. (right) Identification of proteins detected in individual SDS-PAGE bands by Mascot, along with statistical significance of Peptide Mass Fingerprint search (Mascot score) and protein abundance estimation (emPAI).(PDF)Click here for additional data file.S10 FigPurified Hsc20 S34Bpa, Q46Bpa and M51Bpa variants, as indicated, were incubated with purified Ssq1 in the presence of ATP. After UV irradiation (+), or as a control no irradiation (-), reaction mixtures were separated by SDS-PAGE. Migrations of size standard, in kDa, are indicated; Q:J* and Q:J** indicate positions of the Ssq1-Hsc20 crosslinks, which were identified using mass-spectroscopy .(PDF)Click here for additional data file.S11 FigHsc20/Ssq1 recognition is driven by long-range electrostatic forces (a) Electrostatic isopotential contours at +/- 1kT/e  around Hsc20 and Ssq1 and (b) around Hsc20 double substitution variant R37A,R41A. Coloring of structures as in (PDF)Click here for additional data file.S12 FigS. cerevisiae. Phylogenetic relationships among taxonomic groups are depicted on the left.Sequence variability of positions that form the Hsc20/Ssq1 binding interfaces (positions shown in green in (PDF)Click here for additional data file.S13 FigE. coli. Phylogenetic relationships among taxonomic groups are depicted on the left.Sequence variability of positions that form the DnaJ/DnaK binding interfaces (positions shown in green in (PDF)Click here for additional data file.S14 FigMajor clades are indicated.(PDF)Click here for additional data file.S15 FigMajor clades are indicated.(PDF)Click here for additional data file.S16 Figp>0.01 confidence was established as ΔAIC = 7.38 (red dashed line).The co-evolution acceptance threshold with (PDF)Click here for additional data file.S17 Figp>0.01 confidence was established as ΔAIC = 9.473 (red dashed line).The co-evolution acceptance threshold with (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.


Mycobacterium tuberculosis (Mtb) and a few other pathogenic mycobacteria. The PE_PGRS33 protein is among the most studied PE_PGRSs. It is known that the PE domain of PE_PGRS33 is required for the protein translocation through the mycobacterial cell wall, where the PGRS domain remains available for interaction with host receptors. Interaction with Toll like receptor 2 (TLR2) promotes secretion of inflammatory chemokines and cytokines, which are key in the immunopathogenesis of tuberculosis (TB). In this review, we briefly address some key challenges in the development of a TB vaccine and attempt to provide a rationale for the development of new vaccines aimed at fostering a humoral response against Mtb. Using PE_PGRS33 as a model for a surface-exposed antigen, we exploit the availability of current structural data using homology modeling to gather insights on the PGRS domain features. Our study suggests that the PGRS domain of PE_PGRS33 exposes four PGII sandwiches on the outer surface, which, we propose, are directly involved through their loops in the interactions with the host receptors and, as such, are promising targets for a vaccination strategy aimed at inducing a humoral response.PE_PGRS proteins are surface antigens of  Mycobacterium tuberculosis (Mtb), kills approximately two million people every year and latently infects one third of the world’s population. Latency is one of the most remarkable features of TB infection, where Mtb establishes a dynamic equilibrium with the host immune system that lasts for lifetime, with no signs or symptoms of disease [Mtb persists in host tissues mostly in a dormant state. Resuscitation from dormancy, which is orchestrated by a set of cell wall hydrolases [Mtb infection that continuously replenishes the bulk of replicating bacilli after their elimination by the host immune response [Mtb and the host immune response is broken in favor of bacterial replication and tissue damage.Tuberculosis (TB) is still the world’s leading infectious cause of death, according to the World Health Organization (WHO) ,2. Its e disease ,4. It isdrolases ,6,7,8,9,response ,11. TB rMycobacterium bovis Bacille Calmette and Guérin (BCG) is the only vaccine available for TB control [Mtb and the human immune system, paired with the lack of immunological correlates of protection, makes this endeavor challenging [status of vaccine development against TB and highlight the potential role of PE_PGRS proteins, a class of surface proteins with interesting immunomodulatory properties, as vaccine antigens. Given the lack of structural information on PE_PGRS proteins and the importance of structural data to understand protein function, we fill this experimental gap using homology modeling. Using PE_PGRS33 (Rv1818c) as a prototype, this review provides a basis for the unraveling of the functional properties of PE_PGRS proteins as immune modulators.Active TB disease is curable with long-lasting multidrug therapeutic regimens, but the emergence of drug-resistant TB represents a major obstacle to future TB care, with important economic and social consequences . Ambitio control ,14, thou control ,16,17. Tllenging . In thisIn the last two decades, significant progress has been made in the search for a better vaccine against TB ,22,23,24Mtb antigens, to promote early and rapid recruitment of “protective” T cells secreting the Interferon-γ (IFN-γ) at the site of Mtb infection [Mtb and that other functions of the immune responses, perhaps including the somehow neglected humoral arm, are involved in the process. Moreover, certain types of cell-mediated immunity against Mtb are associated with the immunopathology rather than protection, and identifying the T cell immune phenotypes involved, or their Mtb antigen targets, is a still unsolved task [Mtb, suggesting that triggering powerful T cell responses in the human host is part of the Mtb survival strategy [Mtb antigens may promote T cell exhaustion, which impairs host immunity while favoring Mtb replication and tissue damage [Mtb antigens may be potentially deleterious [The main advantage of a whole cell-derived vaccine is the presence of different antigenic components and the possibility to stimulate a broader and diverse immune response than subunit vaccines without the addition of adjuvants, since the mycobacterial cell wall serves as a potent activator of innate immune response . Conversnfection ,26. Howenfection . Mountinved task ,29,30,31strategy ,33. In ae damage . DesigniMtb infections. BCG vaccination is known to induce antibodies, primarily against capsular polysaccharides as arabinomannan and α-glucans [Mtb growth compared to antibodies obtained from active TB patients, thanks to the ability of the former to boost macrophages to kill intracellular Mtb [Mtb antigens as Ag85A and ESAT-6/CFP10, suggesting a potential role of the humoral response in “resisting” infection or at least in preventing the development of overt disease [Mtb infected subjects requires further scrutiny [The role of the humoral response has been questioned for a long time, though recent studies have brought new and compelling evidence for the role of antibodies in -glucans . IgG dir-glucans , and imm-glucans . Hence, -glucans . Antibodular Mtb . A group disease . Hence, scrutiny . CurrentMtb, raising the possibility for new vaccination strategies against TB that target unique surface antigens.Designing new vaccines against TB aimed at eliciting protective antibodies requires a better understanding of the mycobacterial surface antigens, with emphasis on the protein antigens that localize on the outer surface and may be available in the mycobacterial capsule. Among these antigens are proteins belonging to the PE and PPE families, which are unique for virulent mycobacterial species. Some of these proteins have been shown to interact with host components and, as such, are implicated in TB pathogenesis. In this review, we build on structural and functional data to argue that a protein belonging to the PE_PGRS subfamily, PE_PGRS33, may serve as a target for a protective humoral response against Mtb genome coding potential is occupied by unique and peculiar pe and ppe genes, which code for the corresponding protein families [Nearly 10% of the families ,56. PE afamilies : specififamilies , we can families , such asfamilies ,61. Overfamilies .Mtb virulence, in particular in the chronic/persistent phase of the infection [Mtb infection [PE_PGRS localize on the mycobacterial surface ,62,63,64nfection . During nfection ,70, whernfection ,72. Specnfection . Similarnfection ,74, PE_Pnfection  are ablenfection . PE_PGRSnfection . Strong nfection .Mtb-infected animals and TB patients [Mtb killing by activated macrophages; in this case, antibodies may exert their activity since the early steps of Mtb infection. Neutralization of surface antigens may be of relevance also during the late stage of TB pathogenesis, by blocking the interaction of PE_PGRSs with TLRs or other host receptors, thereby reducing the inflammation and tissue damage that is the hallmark of TB disease [Antibodies against PE_PGRS proteins and specifically targeting their PGRS domain have been detected in patients ,78,79. I disease . UnfortuTulasnella calospora, a genus of patch-forming fungi in the Tulasnellaceae family. A conserved linker GRLPI domain (l-GRPLI), likely acting as a transmembrane anchor, connects the PE domain to a distinctive region, not predicted by PFAM, and characterized by multiple repeats containing the GGA-GGX motif interspersed with unique sequences, and commonly denoted as PGRS domain  and EsxB in Staphylococcus aureus.The PE domain takes its name from the conserved Pro–Glu (PE) amino acids at its N-terminus (residues 7–8)  Figure 1Crystal structures have been reported for the two PE domains PE8 and PE25, in both cases forming a heterocomplex with PPE partners . In all By binding PPE, EspG5 protects the aggregation-prone hh motif on PPE proteins and keeps the dimers in a secretion-competent state. Consistently, point mutations of this conserved hh motif affect protein secretion . Both inAs mentioned above, no structural information on the PE domains from PE_PGRS proteins is hitherto known. Neither it is known whether PE_PGRS proteins strictly require a PPE-like domain, as in the case of PE proteins in Mtb encodes for a number of PE- and PPE-containing serine α/β hydrolases, which are possible candidates as PE_PGRS33 hydrolases [M. marinum and PE_PGRS35 in Mtb, is known to cleave the lipase LipY [Importantly, the PE domain of PE_PGRS33 is required for the protein translocation through the mycobacterial cell wall ,88,90,91drolases  . Therefore, we performed homology modeling based on the target–template alignment using ProMod3 and the structure of sfAFP as a template (PDB code 2pne). The alignment of the sequence of this C-terminal PGII module against the entire PGRS region identifies further three modules with the same pattern and sequence identities ranging between 63.0% and 53.9%  of 9.2 Å. This conformation resembles that observed for the polyproline type II (PPII) helices found in collagen [II sandwich, six antiparallel PGII helices are stacked in two antiparallel groups, with three to four triplets spanning the PGII domain length  loops being determinants of host binding. Therefore, similar to PGII of PE_PGRS33, the glycine-rich core of the PGII sandwich of S16 long tail fiber exposes hypervariable β-turn loops that determine receptor specificity  of immunoglobulins [II loops of PE_PGRS33 are likely exposed to the host and, as such, the principal targets of antibodies.In contrast to the antifreeze protein sfAFP, it is likely that in the case of both PE_PGRS33 and the bacteriophage S16 long tail fiber, the flatness of the PGlobulins ,101. As Mycobacterium smegmatis (Ms), in a process mainly governed by its PGRS domain [Mtb cell wall, PE_PGRS33 is in a position that results in the exposure to the milieu and in the capacity to interact with the host [PE_PGRS33 promotes cell death and increases mycobacterial survival in macrophages, as demonstrated by heterologous expression in S domain ,102,103.the host   promotes cell necrosis and inflammation, which increases the survival of the host   of 0.7, suggesting that a biological pressure acts on Mtb to prevent mutations that may impair PE_PGRS33 protein function [II sandwich modules, consisting of tightly packed helices, each made of three or four turns with three residues per turn (the GGX triplets), provides a plastic structure that can tolerate large indels while maintaining proper localization of the unique amino acids that are found in the loops between the helices. PGII sandwiches are typically stabilized by hydrogen bonds involving backbone atoms of glycine residues (N—H-O and C—H-O). Therefore, glycine residues are the sole residues that cannot mutate, as their mutation to any other residues would strongly affect the structural integrity of the PGII sandwich. On the other hand, mutations of non-glycine residues are predicted to hardly contribute to structural stability, as they point toward the external part of the molecule and are not involved in stabilizing intramolecular interactions. Similarly, insertions or deletions of entire triplets, as observed in most frequent alleles  as observed in alleles 48–52  these PGII sandwiches are directly involved through their loops in the interactions with host receptors as TLR2; (c) the sequences encoding these regions result in purifying selection in Mtb, indicating a key role of PGII domains in Mtb survival and TB transmission; and (d) insertions and deletions in the region encoding the PGII domains may have an impact on the immunomodulating activity of PE_PGRS33, as assessed in in vitro and in vivo models. Hence, a strategy aimed at blocking or interfering with the PE_PGRS33–host cell receptor interaction might be useful to block the key steps of TB pathogenesis. As such, a vaccine formulation aimed at eliciting antibodies targeting the surface-exposed loops of PGII domains of PE_PGRS33 may serve to curb inflammatory processes and prevent or reduce the risk of a drift toward necrosis and mounting tissue immunopathology, which is the hallmark of active TB disease.In summary, the observations made and results obtained so far suggest that (a) the PGRS domains of PE_PGRS33 expose four PGMtb surface antigens that are the natural targets of protective humoral responses. In this work, we provide a brief summary of the main challenges associated with the development of a new vaccine against TB and highlight the importance of a structure/function view to investigate the vaccine potential of a unique class of Mtb surface antigens, the PE_PGRS proteins. Among these, PE_PGRS33 can be considered a prototype protein of the family for the availability of experimental data implicating the PGRS domain in the interaction with host receptors. A vaccination strategy aimed at eliciting a protective humoral response shall ideally target secreted or surface-exposed antigens and/or virulence factors that play a key role in microbial pathogenesis. PE_PGRS33 is such a candidate, although the lack of structural data on the peculiar PGRS domain has so far hampered a fine understanding of the proper localization of this protein on the mycomembrane. For years, the glycine-rich PGRS domain has been thought to be highly flexible or unfolded, due to the conformational properties of glycine residues. Using homology modeling, we show that the PGRS portion of PE_PGRS33 is well structured and contains four compact subdomains, named as PGII sandwiches, each consisting of six left-handed PP-II helices stacked in two antiparallel groups. These sandwich domains were previously observed in antifreeze proteins, a class of macromolecules critical to the existence of life at sub-zero temperatures, and in Salmonella phage S16 adhesin. Similar to S16 adhesin, the flat structure of the PGII sandwich domains of PE_PGRS33 may be functional to projecting surface-exposed loops on one side of the molecule, like antigenic loops. Overall, the structural considerations collected here for PGRS domains also allow for the interpretation of the typical polymorphism observed for PGRS domains, since the PGII sandwich domain is able to tolerate modifications, insertions, and deletions, without destabilization. A peculiarity of PGII sandwiches is that they are stabilized solely by backbone hydrogen bonds (N—H-O and C—H-O) involving glycine residues. This feature, with glycine residues pointing inside the fold, where no other residues would fit, explains the high frequency of glycine residues in PGRS domains.Vaccine development against TB has been dominated by strategies aimed at the elicitation of robust T cell responses, while antibody-based strategies have been neglected due also to the limited number and poor knowledge of Therefore, the unique sequences in the PGRS domain coding the surface-exposed loops are likely to be responsible for the interaction with host receptors  and the ideal targets for antibodies with protective potential.II sandwich domains with a humoral response is an out-of-the-box approach to develop new vaccines against TB, taking into account the exposure of multiple copies of PGII sandwiches on the mycobacterial outer membrane.In conclusion, the data examined here provide the structural basis for both the antigenic properties of PGRS domains and of their typical polymorphism. Targeting the surface-exposed loops emerging from the PGMtb. The fact that Mtb expresses more than 50 PE_PGRS proteins on the mycobacterial surface, with structural features similar to those of PE_PGRS33, and that these proteins play unique roles in the Mtb infection process and immunopathogenesis of TB, underscores the potential impact that a vaccination strategy targeting PE_PGRS proteins may have in the control of TB.One important aspect to address concerns the identification of an effective vaccination strategy that can elicit strong, robust, and specific antibody responses against the surface-exposed loops. Inclusion of recombinant PE_PGRS33 purified under native conditions in already available vaccine platforms, such as BCG or new vaccines against TB, may be a strategy that can be useful to pursue . Immuniz