Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from  Microorganisms live in variable environments that require rapid reaction to changing conditions. Consequently, they developed a number of signaling systems that are classified according to the number of distinct molecular entities involved in signal transduction: one-component systems (OCS), two-component systems (TCS), and so on. While OCS usually respond to intracellular stimuli, TCS are able to detect extracytoplasmic molecules by means of a membrane-spanning receptor that transmits the signal inside the cell and controls the activity of its respective soluble response regulator (RR).Due to their versatility and variety of the recognized signals, TCS are widespread among microorganisms: bacterial genomes often encode tens and sometimes more than a hundred receptor and RR genes ,2,3,4,5.Regulation of RR activity is usually achieved via its phosphorylation or dephosphorylation by a cognate histidine kinase (HK) ,24. DepeThere are three major types of TCS, which are based on sensory HKs, chemoreceptors, and sensory rhodopsins. HKs are usually dimeric proteins that perceive the signal and act on RR directly; RRs often regulate transcription and intracellular processes ,24,26. Chistidine kinases, adenylyl cyclases, methyl-accepting proteins, and phosphatases [Per-ARNT-Sim [GMP-specific and stimulated phosphodiesterases, adenylate cyclases, and FhlA [HKs have highly variable modular architectures that, in the minimal form, include the sensor domain, dimerization and histidine phosphotransferase (DHp) domain, and catalytic (CA) domain. They can also contain a transmembrane (TM) domain and additional signal transduction domains  such as phatases ,36,37), ARNT-Sim ,39), andand FhlA ), which and FhlA ,37,41. Sand FhlA ,43. Confand FhlA ,50,51,52and FhlA ,54 or byand FhlA ,56. Finaand FhlA . In geneand FhlA ,59.In this review, we focus on bacterial nitrate- and nitrite-sensing HKs (NSHKs), which are among the best studied TCS. In particular, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information.2O), an ozone-depleting greenhouse gas [Many bacteria can use nitrate or nitrite as an electron acceptor for respiration ,62,63,64ouse gas . Microbiouse gas ,67 and souse gas , and theouse gas . An anaeouse gas .Escherichia coli are prototypical nitrate-responsive HKs, transmembrane proteins that are members of NarQP and NarXL TCSs, respectively [Mycobacterium tuberculosis [Staphylococcus carnosus is a soluble protein with a GAF domain fold that regulates the activity of oxygen-sensing NreBC TCS [Pseudomonas aeruginosa PAO1 [Klebsiella oxytoca (belongs to Gammaproteobacteria) [nitrate- and nitrite-responsive domain [AmiR and NasR transcription antitermination regulators [Consequently, the bacteria need the means to sense nitrates and nitrites and respond to them. Currently, several different microbial nitrate sensors have been identified and characterized. NarQ and NarX from ectively . NarS isrculosis . NreA frreBC TCS . McpN isosa PAO1 . Finallyacteria) , which ce domain ) and ANTgulators ). Interegulators , despiteEscherichia coli [E. coli was discovered, and NarX and NarL were shown to form an HK-RR pair [E. coli genome harbors a second HK involved in nitrate regulation, named NarQ [E. coli genome, narX is adjacent to narL, but narQ and narP are separate from each other and from narXL. However, cross talk between the two systems is also observed (3−), and NarQ responds both to nitrate and nitrite (NO2−), cross talk allows for differential regulation of the output depending on the concentration of the two ions [E. coli. In addition to the nar (nitrate reductase) operon, NarQ and NarX also regulate several other metabolism-related operons such as nir (nitrite reductase), nap (periplasmic nitrate reductase), frd (fumarate reductase), dcu (dicarboxylate uptake), or dms (dimethylsulphoxide reductase) , InterPro contains the information only about the sensor, TM, HAMP, DHp, and CA domains, while the S-helix and GAF-like domains are not recognized (not annotated). Consequently, while searching for NSHKs in InterPro, we obtained the sequences with several architectures with the assumption that some of the other domains may be not recognized as well. We found 4952 sequences with the architecture “sensor, HAMP, DHp, and CA”, 582 sequences with the architecture “sensor, HAMP, GAF, DHp, and CA”, 303 sequences with the architecture “sensor, DHp, and CA”, and 284 with the architecture “sensor, HAMP, and DHp”. These sequences were pooled together into a single set containing 6121 different records, which was used for the downstream analyses.We note that some of the signal transduction domains are very variable and are not always easy to recognize in the sequence. For both Beta- and Gammaproteobacteria, including a subgroup of various sulfur-reducing bacteria , and another contains Bacteroidetes , Caldilinea aerophila [Aggregatilinea lenta [Candidatus Denitrolinea symbiosum [Phylogenetic analysis reveals that the obtained sequences form several large and several smaller clusters as well as some isolated sequences. NarQ and NarX are grouped with the genes from bacteria . Two othroidetes . Finallyerophila , and Aggea lenta , as wellea lenta ,114,115 ymbiosum . Escherichia coli, Pseudomonas aeruginosa, endosymbiont of Riftia pachyptila, Thiobacillus denitrificans, Neisseria meningitidis, Paraburkholderia graminis, Burkholderia pseudomallei, Aggregatilinea lenta, Haemophilus influenzae, Vibrio cholerae, Joostella marina, Indibacter alkaliphilus, and Thermoflexibacter ruber. We observe that almost all of these genes contain other nitrate/nitrite-related genes in the vicinity , supporE. coli genome contains two NSHK genes, narQ and narX, with amino acid identity of ≈29.4%. We used CD-HIT [Enterobacterales, although examples from other bacterial orders are also observed , GAF-like, DHp, and CA  NarQ-like proteins from Enterobacterales, Pasteurellales, Vibrionales, and Alteromonadales, clustering close to E. coli narQ; and (iii) NarX-like proteins: all other NSHKs.Multiple sequence alignment reveals remarkable conservation of the overall features of IPR029095-containing proteins. Most sequences are encoding full-length proteins with all of the seven domains intact. Yet, in some of the proteins, the region between the TM helices and the DHp domain is shortened. For example, in oli narQ  but not  cluster . Careful α-helix . Based o α-helix  and arch α-helix , we suggGiven the high level of the overall amino acid sequence conservation, including the nitrate-binding motif and the residues important for phosphorylation, we presume that all these sequences encode mostly functional NSHKs. We analyzed how well the individual domains are conserved and found notable variation. Sensor and HAMP domains generally vary to the same degree as the whole protein; TM α-helices and the region corresponding to the GAF-like domain are less conserved, whereas the catalytic domain and especially S-helix and DHp are conserved better than the protein overall .Most sensor HKs are complex dynamic multi-domain proteins. While full-length structures of several soluble sensor HKs have been determined previously ,133, no In the absence of full-length structures of TCS, a divide-and-conquer approach has been very fruitful: structures of different domains are determined individually, and then, the model of the whole protein is assembled from parts. In particular, a plethora of sensor domain structures are known at the moment that highlight many different modes of ligand binding: symmetric, asymmetric, at the dimerization interface, and reveal signaling-associated conformational changes ,42,138. Below, we review what is known about each NSHK domain. NSHKs, having seven domains, are more complex compared to many other HKs. Experimentally determined structures are currently available for the sensor, TM, and HAMP domains ,140,141.The sensor domain of NSHKs is responsible for binding the ligand (nitrate or nitrite) and transmitting the signal downstream. It is located in the extracytoplasmic space, preceded and followed by TM α-helices . EarlierE. coli NarQ and NarX . Evidently, the P-box is extremely well conserved among NSHKs. The nitrate-binding amino acids, forming the motif G-x-x-R . This fact underlies the importance of the respective junctions for signal transduction and allows us to analyze the amino acid frequencies in the TM region . Overallignaling , and simse, PhoQ ,163. The HAMP domain is a previously enigmatic module often found in the cytoplasmic part of HKs, chemoreceptors, and sensory rhodopsin transducers adjacent to the TM domain ,37,164. Several mechanisms of HAMP domain signaling have been proposed, namely diagonal scissoring, helical rotation, and transitions between stable (compact) and dynamic states ,37,176 aBacteroidetes NSHKs appear to lack HAMP domains altogether , with the characteristic motif L-E-x-x-V-x-E-(R/K)-T-x-(E/D/Q)-L ,185. Sinoti DctB  was crysoti DctB ,190. Whioti DctB ,192) thaoti DctB ,193,194.90-E83-Q20-R46-V83-A20-E45-K76-T86-A30-E18-L82, where the subscripts indicate the probability of observing the respective amino acid at this position (in percent). Modeling shows that there are two possible arrangements . In the phosphatase state, the domain catalyzes dephosphorylation of the RR. DHp domains determine the specificity of HK:RR interactions ,207,208.Based on their sequences, DHp domains are grouped into several subfamilies . All NSHtrans or in cis [Many structures of DHp domains are currently available ,25,41,58r in cis . Howeverr in cis .Bacillus subtilis DesK has been extensively characterized, and multiple X-ray structures in kinase and phosphatase states are available [E. coli NarQ and NarX DHp domains is 29% and 26%, respectively. The regulation of DHp activity is achieved via the conformation of the first α-helix and the conserved histidine, which are controlled by the preceding domain  [Whereas experimental structures of any NSHK DHp domain are not available at the moment, the structures of another member of the HisKA_3 subfamily that have been determined previously can be used as a model. vailable ,215,216;l NSHKs) .Catalytic domains of histidine kinases catalyze transfer of the γ-phosphate group from ATP to the catalytic histidine of the DHp domain. They are a part of a larger protein superfamily comprising proteins similar to DNA gyrase B, topoisomerase, heat shock protein HSP90, phytochrome-like ATPases, and DNA mismatch repair proteins . E. coli NarQ CA harbors two cysteine residues in close vicinity to each other, Cys455 and Cys494 (Enterobacterales and Pasteurellales proteins; Cys494 is relatively well conserved among Enterobacterales and Vibrionales proteins. In an unrelated HK SrrB, similar cysteines, Cys464 and Cys501, form an intramolecular disulfide bond, which responds to the cellular redox environment and affects autophosphorylation kinetics [The activity of the catalytic domain is likely regulated not by itself but rather by the DHp domain, assuming a conformation that is more or less conducive to phosphorylation depending on the signaling state of the protein. Yet, there is another interesting possibility in some of the NSHKs. Opposite to the catalytic site, d Cys494 . Cys455 kinetics . Thus, ikinetics  is achiekinetics ,221, or kinetics .What is the structure of full-length NSHKs in a native environment, in ligand-free and ligand-bound forms?How dynamic are full-length NSHKs? How many different conformations are assumed by NSHKs in each signaling state? How are these correlated with interactions with response regulator proteins?What is the mechanism of nitrate–nitrite discrimination by NSHKs at the molecular level? How does oxygen and/or the cellular redox environment affect the activity of NSHKs? Are there any other factors that affect the activity of NSHKs?Bacteroidetes? How does the absence or presence of the HAMP domain influence signal transduction in NSHKs?What is the role of the GAF-like domains, and how is the signal transduced in GAF-less NSHKs from What is the role of NSHKs in all the different organisms?With this review, we attempted to summarize the knowledge about nitrate-responsive histidine kinases. It is apparent that they are important proteins for many microorganisms, and they have been studied accordingly. We know how and when they are activated and which cellular processes they regulate. Yet, many questions remain:How many different TCS sensors may be found in nature? What kind of signals and environmental factors may be recognized?Are the signal transduction mechanisms conserved between different HKs? Between HKs and chemoreceptors?What are the design principles of sensor HKs? How sensor HKs may be efficiently employed in the generation of new molecular biology tools, including artificial sensors ,17,18 anThe information about the signaling mechanisms of NSHKs may be useful in the studies of other HKs as well and vice versa. The following questions go beyond the intricacies of nitrate regulation in microorganisms:Given the number of microorganisms and abundance of signaling systems in each of them, answering all of these questions will not be easy. However, it is clear that the advances in the available molecular biology techniques, and development of new ones, will bring deep insights intro microbial signaling and its applications.


Binding to plasminogen (Plg) enables bacteria to associate with and invade host tissues. The cell wall protein PbsP significantly contributes to the ability of group B streptococci, a frequent cause of invasive infection, to bind Plg. Here we sought to identify the molecular regions involved in the interactions between Plg and PbsP. The K4 Kringle domain of the Plg molecule was required for binding of Plg to whole PbsP and to a PbsP fragment encompassing a region rich in methionine and lysine (MK-rich domain). These interactions were inhibited by free L-lysine, indicating the involvement of lysine binding sites in the Plg molecule. However, mutation to alanine of all lysine residues in the MK-rich domain did not decrease its ability to bind Plg. Collectively, our data identify a novel bacterial sequence that can interact with lysine binding sites in the Plg molecule. Notably, such binding did not require the presence of lysine or other positively charged amino acids in the bacterial receptor. These data may be useful for developing alternative therapeutic strategies aimed at blocking interactions between group B streptococci and Plg. A wide variety of bacterial species are capable of interacting with plasminogen (Plg), a process that is thought to enhance their ability to colonize and invade host tissues . Plg is via their lysine-binding sites (LBS) with multiple ligands, including host Plg receptors/targets, such as fibrin and α2 anti-plasmin, and bacterial proteins  bears an Activation Peptide (AP) domain followed by 5 Kringle domains  and by the S1 Peptidase Domain (SPD) at the C-terminal region . The Kriproteins . The Kriproteins . LBS in proteins  while thvate Plg . Other evate Plg . Bacterivate Plg  as well vate Plg . A commoStreptococcus agalactiae  is a gram-positive encapsulated bacterium that can behave as a commensal of the human gastrointestinal and genital tracts or as an agent of invasive infections. Clinical manifestations of GBS disease include sepsis and meningitis in neonates and an increasing variety of conditions in adults with predisposing factors, in pregnant women and in the elderly (Plasminogen binding surface Protein), a cell-wall-anchored protein of 521-aa. PbsP contains an N-terminal domain, two 150-aa SSURE repeat domains , SSURE domains (rSSURE-1+2 and rSSURE-2) and rMK-rich domain used in this study were produced as described . The pepE. coli BL21 (DE3) strains were grown to purify the peptides as fusions to Gluthatione S-Transferase, as previously described  was obtained by digestion of Plg with porcine pancreatic elastase (Sigma-Aldrich), as previously described . After centrifugation, the plasma fraction was frozen in aliquots and stored at −20°C . Plasminescribed .Binding of Plg or its fragments to recombinant proteins  was determined by ELISA, dot and Western blot assays as described . For ELIIn order to identify the molecular regions involved in interactions between PbsP and Plg, we analyzed the binding of isolated recombinant PbsP domains to different Plg fragments by ELISA. In these assays we used the whole PbsP protein, the MK-rich domain and 2 different SSURE domain fragments  spanning the remaining part of the domain  lacking the 25 N-terminal amino acids and Fr3, the complementary fragment, spanning the 25 N-terminal amino acids (S444-A468). As shown in 423 to T443) of the MK-rich region are not required for Plg binding and that Fr1, but not shorter fragments, recapitulates the ability of the MK-rich domain to bind Plg.In view of the significant Plg-binding activities of the MK-rich domain of PbsP, further studies focused on the mechanisms of such interactions. In order to identify the minimal region of the MK-rich domain that still maintains reactivity with Plg, we produced truncated forms of this domain. First we produced an N-terminally truncated form (designated Fr1) spanning amino acids SPrevious studies have demonstrated that terminal or internal lysine residues in a number of bacterial Plg receptors are required for optimal interactions with Plg . In viewA remarkable feature of the MK-rich region is the abundant presence of methionine, which accounts for 26% of all amino acid residues  of PbsP involved in binding to Plg. In agreement with previous studies , we founresidues . TherefoIn conclusion, we identified here a novel bacterial sequence that can interact with the LBS of Plg even in the absence of lysine or other positively charged amino acids. These data may be useful to devise alternative therapeutic strategies to prevent Plg-mediated invasion of host tissues by GBS and other bacterial pathogens.The original contributions presented in the study are included in the article/FC, LR and GP conducted most of the experiments and analyzed data. Other experiments were conducted by GL, GG and RG. CB and GT contributed to the study design, wrote the paper and revised the final version of the manuscript. All authors contributed to the article and approved the submitted version.The work was funded in part by the PRIN  grant 2017M8R7N9_002 from the Ministero dell’Università and Ricerca Scientifica (MIUR) of Italy and by FFABR (Fund For Basic Research Activities) 2017 and 2019 from the University of Messina. Additional funding was provided by a grant for PhD student (PON n° DR_36_TRAN_PON_IND_1) at Doctoral School in Translational Molecular Medicine and Surgery, Department of Biomedical, Dental and Imaging Sciences, University of Messina, Messina, Italy.We thank Dr. Patrick Trieu-Cuot from Institute Pasteur, Paris, France, for helpful discussion and suggestions.CB and GT act as scientific advisors for, respectively, Scylla Biotech Srl. and Charybdis Vaccines Srl. without receiving any compensation for these activities. Charybdis Vaccines S.r.l. and Scylla Biotech S.r.l. did not provide funding for this study and had no role in its conduction.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SUV39H1 and SUV39H2 were the first protein lysine methyltransferases that were identified more than 20 years ago. Both enzymes introduce di- and trimethylation at histone H3 lysine 9 (H3K9) and have important roles in the maintenance of heterochromatin and gene repression. They consist of a catalytically active SET domain and a chromodomain, which binds H3K9me2/3 and has roles in enzyme targeting and regulation. The heterochromatic targeting of SUV39H enzymes is further enhanced by the interaction with HP1 proteins and repeat-associated RNA. SUV39H1 and SUV39H2 recognize an RKST motif with additional residues on both sides, mainly K4 in the case of SUV39H1 and G12 in the case of SUV39H2. Both SUV39H enzymes methylate different non-histone proteins including RAG2, DOT1L, SET8 and HupB in the case of SUV39H1 and LSD1 in the case of SUV39H2. Both enzymes are expressed in embryonic cells and have broad expression profiles in the adult body. SUV39H1 shows little tissue preference except thymus, while SUV39H2 is more highly expressed in the brain, testis and thymus. Both enzymes are connected to cancer, having oncogenic or tumor-suppressive roles depending on the tumor type. In addition, SUV39H2 has roles in the brain during early neurodevelopment. The unstructured N-terminal tails of the histone proteins protrude from the core nucleosome and contain complex patterns of post-translational modifications (PTMs), including the methylation of lysine and arginine residues, lysine acetylation and the phosphorylation of serine and threonine ,2,3,4. TSUV39H1 and SUV39H2 consist of two-conserved domains, one SET- and one chromodomain . The amiOne large group of PKMTs contains a SET domain as the catalytically active part, which consists of approximately 130 amino acids ,18,23. TThe SET domain of SUV39H2  has beenSUV39H1 is able to introduce trimethylation at H3K9 in vitro, but the conversion of H3K9me2 into H3K9me3 is slow ,24,25. TSimilar to SUV39H1, SUV39H2 introduces H3K9me3 in vitro ,32. It pInterestingly, the substrate specificity profiles of the two SUV39H enzymes differ from each other . OverallS. pombe, automethylation was observed on K455 and K472, which are located in an autoregulatory loop (ARL) positioned between the SET and post-SET domain [In Clr4, the SUV39H homolog in T domain . This ART domain ,37, whicT domain , has beeT domain , suggestChromodomains are well-known methyllysine interaction domains ,21. StruBiochemical studies revealed that the chromodomain of SUV39H1 inhibits its methyltransferase activity, and this inhibition was relieved by H3K9me3 binding to the chromodomain [In 2017, two additional papers shed more light on the targeting and regulatory role of the SUV39H1 chromodomain ,41. CollThe targeting and regulation of SUV39H1 by RNA binding to its chromodomain is also consistent with the finding that the telomeric TERRA RNA associates with this domain and this interaction promotes the accumulation of H3K9me3 at damaged telomeres and end-to-end chromosome fusions . CurrentThe chromodomains of HP1 proteins are critical readers of pericentromeric H3K9me3 ,44. StriThe expression profiles of SUV39H1 and SUV39H2 in mice are overlapping during embryogenesis, but SUV39H2 remained expressed in adult testis where it is localized at meiotic heterochromatin . In humaAs mentioned above, H3K9me3 is a key feature of constitutive heterochromatin in eukaryotes ,8,9,10 aSUV39H1 and SUV39H2 also contribute to the chromatin modification network via different pathways, because HP1 proteins also recruit SUV4-20H enzymes to heterochromatic regions, where they generate H4K20me3 by using the H4K20me1 provided by SET8 as a substrate ,58,59. BAnother poorly understood observation is that SUV39H1 exists in multimeric complexes with the other H3K9 PKMTs such as G9a, GLP and SETDB1 C and theAs described above, the specificity profile of SUV39H1 differs from SUV39H2, suggesting that these paralogs could have non-redundant functions in the methylation of non-histone proteins. Based on the specificity profile, several SUV39H1 non-histone substrates were identified . The metOther non-histone substrates of SUV39H1 also have chromatin associated roles: The methylation of RAG2 by SUV39H1 occurs within its NLS and it was shown to alter its sub-nuclear localization . This obDOT1L is an evolutionarily conserved 7-beta-strand histone PKMT specific for lysine 79 of H3 (H3K79), which has important roles in development and cancer . DOT1L-dFurthermore, SUV39H1 has been connected to immune function and bacterial infections, because the mycobacterial histone-like HupB protein has been shown to be methylated by SUV39H1 and this process participates in host defense . The SUVSUV39H2 was found to trimethylate LSD1 at K322  creatingAs described above, SUV39H1 and H3K9me3 are predominately associated with the generation and maintenance of constitutive heterochromatin. In mammals, defective pericentric heterochromatin and aberrant transcription of pericentric repeats are associated with genomic instability and cancer ,74. ThesHuman SUV39H1 has been implicated in a variety of complex biological processes such as DNA damage repair ,76,77, t1 cell cycle arrest [Accumulating evidence indicates that SUV39H2 acts mainly as an oncogene that contributes to the initiation and progression of cancers including invasion and metastasis . As mente arrest .In concordance with its high expression in cerebellum, SUV39H2 has also been connected with neuronal effects. It has been shown that stress-induced H3K9 methylation in the hippocampus was correlated with an upregulation of SUV39H2, suggesting that the enzyme plays a functional role in this process . RecentlMoreover, the SUV39H2 N324K loss of function mutation  has beenThe biological function of a PKMT is intimately connected to its substrates and the changes of the substrates’ properties associated with target lysine methylation. For SUV39H1 and SUV39H2, H3K9 is a main substrate. It needs to be studied systematically, how intrinsic and external signals lead to changes in H3K9me3 levels in the heterochromatin and at defined genomic target loci. Moreover, both enzymes also methylate non-histone proteins. The identification of more non-histone proteins and deeper knowledge about their roles in the cellular signaling network will be important for the complete understanding of the biological role of SUV39H1 and SUV39H2. Combined achievements in both directions will help to understand the roles of SUV39H enzymes in diseases better and they may result in novel and specific therapeutic strategies.


Jump performance was assessed using MyJump2, a scientifically validated mobile phone app. Intergroup and intragroup comparisons were assessed, and the magnitude of change was calculated using the effect size (ES). While CG significantly decreased the relative power over time , results from the intragroup comparisons suggest that IG significantly increased the countermovement jump (CMJ) height , the relative force , maximal velocity , and relative power . We concluded that a 16-week strength training program of lower limbs is an effective way to improve CMJ height in young dancers. Supplementary strength training appears to be the determinant for the improvement of the jumping performance of ballet dancers.Jumping ability is considered a determinant of performance success. It is identified as one of the predictors and talent identification in many sports and dance. This study aimed to investigate the effect of 16 weeks of lower-limb strength training on the jumping performance of ballet dancers. A total of 24 participants from the same dance school were randomly selected in the control group [CG;  Both sports and dance culminate in public performances. The intersections of dance and sports are reported since some athletes and coaches use strength training to improve their performance , particugrand jeté leap performance in dancers with different skill levels, being considered useful tools for monitoring the power-generating capacity of the lower body of the dancer, thus giving insight into the overall jumping capacity of the dancer  . In facte dancer . Conseque dancer .petit and grand allegro exercises at the beginning of center work, for 8 weeks, as an intervention to analyze jumping ability  evaluated the mechanical variables during the CMJ of dancers, (2) proposed a specific training program, for 16 weeks, to improve the jumping performance, and (3) compared the mechanical variables during the CMJ of dancers before and after a specific jump training program application. We hypothesized that the proposed training program will positively affect the CMJ height of the dancers.The following sample inclusion criteria were established: (1) enrollment at the educational institution participating in the study; (2) absence of injury that prevented them from training in the last 3 months, during the intervention and at the time of evaluation; (3) no involvement in any complementary physical training or sports activity during the 16 weeks of intervention; and (4) attendance at a minimum of 80% of training sessions.n = 10) and the IG (n = 14). Although the participants were selected randomly, the groups did not have the same number of subjects because four dancers from the CG were involved in a complementary sports activity, which led to their exclusion from the CG sample. The sample size was restricted to the number of higher-level dancers from the dance school that accepted and approved to carry out this study.Our sample was composed of 24 ballet dancers from the same school dance and was divided into the CG  years, 5 years of regular dance practice, and 28.50 h of training per week . The IG All ethical procedures were carried out following the Declaration of Helsinki, and the study was approved by the local Ethical Committee (CE/FCDEF-UC/00742021). All participants and their guardians (dancers aged under 18 years) were informed of the benefits and risks of the research. Before the beginning of the study, they signed an informed consent document for testing, implementation of the training protocol, and publication of collected data.2), CMJ (cm), relative force (N/kg), maximal velocity (m/s), relative power (W/kg), and the height of push-off (HPO) (cm) of each dancer. The relative values to body mass were calculated by dividing the output of each subject by their mass as previous findings, whereas relative force was calculated by dividing the force output of each subject by their mass (The variables assessed in this investigation were the weight (kg), height (m), body mass index (BMI)  and the stature (m) were collected using a Tanita SC-330  and an aluminum stadiometer , respectively. HPO assessment , 2014 imBefore all control time points, dancers performed their habitual warm-up routine consisting of 15 min of jogging, dynamic stretching , and preparatory CMJs, also following previous orientations . Before Both groups (IG and CG) maintained the standardized training regimen  to include a data collection performed at the 8th week only at the IG as a control measure of jump performance evolution. The level of significance was set at p ≤ 0.05.All data are presented as means (SDs) using IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY, United States. The normal distribution of the study variables was assessed using the Shapiro–Wilk test. Intergroup and intragroup comparisons were evaluated by an independent measure and a repeated measures via magnitude-based qualitative inferences  were found between the sexes in the jumping performance. Accordingly, we considered them as one group for the study analysis. Although our sample is composed of female and male dancers, similar to a previous study , no signp = 0.039, ES = 0.911: large), relative force , maximal velocity , and relative power values  in comparison to the IG. In posttest, no significant differences were found between groups in any of the study variables.In pretest, the CG presented significantly higher CMJ (p < 0.001). In contrast, the IG significantly increased all variables, namely, the anthropometric measurements, weight , height  and BMI , CMJ , relative force , maximal velocity , and relative power .The CG significantly decreased relative power values over time (p = 0.002). Regarding the jumping performance, CMJ (p < 0.001), maximal velocity (p < 0.001), and relative power (p < 0.001) increased significantly and progressively across all evaluation moments. Finally, relative force increased significantly between moments 1–2 and 1–3 (p < 0.001).Finally, p < 0.001), intragroup comparisons indicate that the IG significantly increased the CMJ height , relative force , maximal velocity , and relative power  after the 16-week training program. The ES interpretation and the individual response results suggest that these improvements in CMJ, relative force, velocity, and relative power represent almost certainly (100%) a positive effect from the training program , relative force , maximal velocity , and relative power values  in comparison to the IG. Regarding the CMJ, this advantage of the CG over the IG in pretest  with a large ES is in opposition with the previous findings where the IG presented an initial advantage in this variable  pre-post 30 weeks of a plyometric training program and in CMJ with arm swing dancers jumped 21.5 (3) cm vs. 25 (2.8) cm , an ES equal to our findings in CMJ  cm and 18.9 (2.7) cm  and relative power (18.14 (1.96) W/kg vs. 20.99 (2.01) W/kg, p < 0.001, ES = 1.37: large), and since our sample was composed of younger dancers, it was important to calculate these variables relative to body mass of each dancer from simple computation measures based on body mass, jump height (from flight time), and push-off distance  m/s vs. 2.31 (0.13) m/s in dancers (Regarding the significant improvements in the IG relative force (16.38 (1.07) N/kg vs. 17.36 (1.09) N/kg, distance . A previdistance . We obsedistance  and valudistance . Althoug dancers  and 2.33 dancers . Previou dancers .p ≥ 0.05). The apparent sample homogeneity could be explained by the maturation process, whereas girls could be at a higher maturity stage and balanced the performance of the boys. Accordingly, we considered them as one group for the study analysis.As stated previously, we controlled the growth of the dancers over the 16 weeks of training using HPO measurements. Still, younger dancers are inevitably in maturation and growing processes, similar to previous studies , which mSince we could not measure the jumping performance every 3 weeks during the program, and 1 week after the end of the program as recommended , the addp < 0.001, ES = − 0.29: small) during the 16 weeks with standard ballet classes. These findings clearly correspond to previous research suggesting that dance training alone may not provide sufficient overload to evoke a physiological change in adolescent dancers and identified that the inclusion of a strength training program facilitated improvements in maximum lower-body strength  is aligned with previous outcomes that also conducted additional evaluation moments  . It woulThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee, Faculty of Sports Science and Physical Education, University of Coimbra (CE/FCDEF-UC/00742021). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.LÁ-C, FC, JE-Á, and LR participated in study design, data collection, and the writing of the first draft manuscript. LÁ-C, FC, JE-Á, BG, IL, and LR participated in the article collection and analysis. LÁ-C, FC, JE-Á, IL, and LR participated in the writing of the methodology and results, and the final revisions of the manuscript. All authors have read and approved the final version of the manuscript and agreed with the order of presentation of the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enterobacteria and Staphylococcus aureus were not detected during storage. Sensory attributes throughout storage were very similar for all high-fat sausages; the odor in the formulations was defined as “different” but not unpleasant. The low-fat sausages, defined as a new product different from conventional sausages, were well accepted by the panelist. Banana flours are a suitable ingredient option to add nutritional value to Frankfurter-type sausages, which can be consumed by the wheat allergic population.This study aimed to develop a fiber-enriched Frankfurter-type sausage by incorporating underutilized green banana flours as a meat extender, replacing wheat flour with banana flours (8%). A low-fat formulation substituting 12% pork fat with 24% banana peel flour was also studied. Sausages were stored at 4 °C/15 days. Cooking loss was low (5.6–4.1%) in all formulations and the substitution of wheat flour with banana flour did not modify moisture and protein composition, while carbohydrate, fiber, and ashes varied with the flour composition. In the low-fat sausages, fiber carbohydrate and ashes increased the most. Texture and color parameters were very similar for high-fat sausages throughout storage, although low-fat sausage showed higher hardness, while chewiness, L*, and whiteness tended to decrease. During the first week of storage, the microbial growth was scarce and then, an increase, except in the low-fat batch, in which growth remained constant.  Recently, the meat industry has focused on developing healthier products due to consumer demands for high nutritional value products with balanced formulations. One of the strategies followed by the industry is foodstuff reformulation, adding ingredients that improve health and limiting undesirable components ,2,3. In There are different possibilities to achieve healthier meat products, including the addition of ingredients such as fibers or resistant starch as binders that would substantially improve the product ,10. In tThe banana crop is one of the main resources in the economy of several South American countries, like Ecuador, being the banana world production ~113 million tons . Banana The present study aims to evaluate the effect of the inclusion of different green banana flours from the pulp, peel, or the whole banana, on the technological, physicochemical, microbiological, and sensory properties of cooked gel-emulsion sausages/Frankfurter-type sausages. Besides, the use of banana peel flour as a fat replacer in the formulation of sausages will be evaluated. The evolution of the sausages during 15 days of chilled storage was also studied.Musa acuminata AAA) were purchased in a local market of Ambato (Ecuador). The fruits were selected according to homogeneous green color and uniformity. The bananas were thoroughly washed in water, and after that, they undergo different processes depending on the flour to be obtained: bananas were cut into slices ~0.03 cm thick (whole banana) or skinned and cut into slices ~0.03 cm thick (pulp). The peel resulting from the process to obtain the pulp was cut with a length of ~0.5 cm (peel). All batches were uniformly spread out in trays to be dried in a convective dryer (Gander MTN) at 60 °C until reaching the constant moisture (around 12%). Later, all the dried material of each batch was crushed in an industrial mill , and the flours obtained were: whole banana flour, banana pulp flour, and banana peel flour.The green bananas , banana pulp flour , peel banana flour . Wheat flour  was used for control purposes.Frankfurter-type sausages were produced according to a standard procedure, where the control formulation (Control) is composed of meat protein (31% beef and 27% pork), 15% pork-back fat, 9% of ice, 8% of wheat flour, and 6% of seasoning . Three types of new formulations were made changing the wheat flour in the same percentage (8%) by whole banana flour (HF-WBF), banana pulp flour (HF-PUBF), or banana peel flour (HF-PEBF); the rest of the ingredients were the same in all batches . A low-fat, fiber-enriched formulation was also elaborated, reducing the incorporation of pork back fat at 3% (instead 15%) and substituting the 8% of wheat flour by 24% of banana peel flour to obtain a product of potential nutritional interest with respect to its fat and fiber content (low-fat fiber-enriched sausages); the other ingredients were not modified, constituting the batch LF-PEBF.For sausages production, meats were ground in a mincer (Mainca PM-21 Spain) for all batches. Then the minced meat, fat, and 25% of crushed ice were homogenized in a cutter (Mainca CM-21 Spain) for 2 min, and subsequently, the seasoning and 25% of crushed ice were added and homogenized 2 min; finally, each flour and 50% crushed ice was added in the corresponding formulas, homogenizing 1 min. Each final batter was stuffed into an artificial Viscofan casing (16 mm diameter/150 mm length). Later, sausages were cooked in a water bath at 80–85 °C until the core of the product reaches 73 °C (~20 min). After heating, the sausages were cooled in a cold bath until an internal temperature of 30 °C. Finally, sausages were packed in high-density polyethylene plastic food bags and refrigerated at 4 °C for 15 days. The elaboration of the sausages was conducted in triplicate (three independent experiments).The yield loss in processing was determined during heat treatment; for that, the Frankfurter-type sausages were cooked (amounts of approximately 1 kg) and weighed before and after cooking. Cooking loss, expressed as a percentage, was evaluated as the initial weight before cooking minus the final weight after cooking. The results are expressed at least by triplicate.Proximate composition  was evaluated following the official methods AOAC 19 927.05, AOAC 923.03, AOAC 2001.11, and AOAC 2033.06, respectively  1/2. For the calibration of the colorimeter, before measurements were done, the white tile standard was used. At least 15 measurements were performed in different areas of the samples. Analyses were performed at 1, 3, 6, 9, 12, and 15 days of chilled storage.Enterobacteriaceae on a double layer of Violet Red Bile Glucose Agar (VRBG)  incubated at 30 °C/72 h; and Staphylococcus aureus on Baird Parker agar (Difco) supplemented with egg yolk tellurite were incubated at 30 °C for 48 h. All analyses were performed in triplicate. The test was carried out on day 1, 4, 8, 12, and 15 days of chilled storage.Ten grams of Frankfurter-type sausages were aseptically placed into a sterile stomacher bag. They were then homogenized with 90 mL of buffered 0.1% peptone water  in a stomacher homogenizer  for 1 min at room temperature. For each sample, appropriate serial decimal dilutions were prepared in peptone water. Mesophilic aerobic bacteria were determined in pour plates of PCA agar (Difco) incubated at 30 °C for 72 h; Mold and yeast were spread plated on Rose Bengal Agar (RBC) (Difco) and incubated at 25 °C; Fifteen semi-trained panelists assessed attributes such as smell, taste, and overall acceptability of the sausages. For this, the panel received training in previous sessions. A sensory acceptance test was performed using a 5-point hedonic scale . Two cylinders (1.6 cm in diameter × 3 cm length) of grilled samples without casing were immediately given to the panelist for evaluation. A glass of water and salted crackers for a palate-cleansing were also provided. Analyses were carried out on day 1, 3, 6, 9 12, and 15 days of chilled storage.The data were processed and analyzed with the GraphPad Prism 5.0 program . Analysis of variance was performed using the one-way or two-way ANOVA test, and when this was significant, the comparison of the means was performed using the Tukey test (α = 0.05).p < 0.05) even though none differ more than 1% from the Control . Frankfurter-type sausages are considered to be a high-intermediate moisture food, with values oscillating between 50–75% [The proximal composition of the different sausage formulations is shown in n 50–75% . In the n 50–75% , studyinn 50–75% .p < 0.05) (The ash content was different in most of the sausages ( < 0.05) ; these dDespite the significant differences in protein content in all batches, the values did not differ by more than 0.59% compared with the control sausage .These results were similar to those presented by Choe and Kim  in sausap < 0.05) in the fat content of the sausages (p < 0.05). Some authors also observed cooking losses, mainly of fat, when the formulation contained fibers as partial fat substitutes [The substitution of wheat flour (Control) with banana flours  at equal proportions showed a very slight decrease  (p < 0.05) as compared with the rest of the formulas with the same amount of flour . In agreement with nutritional claims, the high-fat sausage formulations could be considered as a “source of fiber” since all of them have more than 3% fiber (3 g fiber/100 g of product). Moreover, sausages including banana peel flour, both high and low in fat , could be labeled as “high fiber” products because both of them contain more than 6% fiber [The fiber content of the sausages depends directly on the composition and concentration of flours used in the formulation ( < 0.05) . Thus, a6% fiber . In meat6% fiber  or banan6% fiber .p < 0.05)  . These rp < 0.01); HF-WBF and HF-PUBF registered a lower caloric content compared to the Control . However, despite having the same proportion of flour, HF-PEBF showed a lower caloric content (11%) probably due to the lower amount of carbohydrates and fat of this formulation. In addition, the low-fat, high-fiber sausages contained 24% less energy   . These sp < 0.05)  . Accordip < 0.05). Differences among lots depended on the type of flour used in the formulations. Compared to the Control, hardness was higher for HF-PUBF and lower for HF-PEBF, while the values were the same for HF-WBF (p < 0.05), attributed to the high fiber content as reported by Atashkar et al. [p < 0.05) on the first day of storage, increasing variability throughout chilled storage and reaching similar values to the initial one after 9 days. In beef emulsion modeling systems prepared with tropical flours (breadfruit or banana) at different inclusion levels , Huang and Bohrer [p <0.05), which showed higher values during the first days of storage (p < 0.05), probably due to the lower fat content and the high fiber and carbohydrate contents that could make this batch less chewy. Regarding adhesiveness, the initial values of the Control and HF-PUBF were higher than those of the rest of the formulations (p < 0.05) but later they declined, while in the rest of the lots the values remained stable. Some studies on fat replacement with different kinds of flour or fiber have been reported in the literature, but it is difficult to compare their effects on the formulations considering the great variability and differences in the fat substitutes used (type and quantity), as well as in the possible variation of water or protein contents in the formulations. In short, in the present work, the substitution of fat with any other ingredient or modification in the formula is intended to change the texture parameters as little as possible to resemble the traditional product (Control). The water immobilized by the hydrocolloids and the water available to react with meat proteins during the emulsification process should be balanced [The replacement of wheat flour with banana flours in the high-fat formulation led to slight modifications in sausage hardness . However, it declined with the incorporation of banana peel flour, being more pronounced in the reduced-fat formulation (LF-PEBF), where the sausages acquired a darker color.The influence of green banana flour in the color of Frankfurter-type sausages is shown in p < 0.05). Intermediate values corresponded to the batch with the lowest fat content (LF-PEBF), while the greatest values for redness were achieved for HF-PUBF due to the presence of banana pulp, which may show some Maillard reaction pigmentation produced during the drying process. Throughout storage, redness exhibited some fluctuations with intermediate values, however, at the end of storage similar values between 4.5–5 were reached in all batches.Initially, redness (a*) levels were similar for the Control, HF-PEBF, and HF-WBF (p < 0.05). During chilled storage, all lots suffered slight oscillations, although there were two clear tendencies, LF-PEBF and HF-PEBF showed higher values, while HF-PUBF and HF-WBF presented the lowest, and the Control showed intermediate values; except for the last day when the values of almost all lots were the same (p < 0.05). This implies that banana peel is the main contributor to yellowness, while the presence of pulp flour favors its decrease.Regarding yellowness (b*), the Control showed the most pronounced initial values, while the HF-WBF and LF-PEBF exhibited the lowest . The most notable were the lower hue and chroma in HF-PUBF, chroma being also low in HF-WBF.Chroma and hue in general showed a quite stable tendency despite slight oscillations during the period studied  until reaching around 5.4 log CFU/g C , counts being significantly lower in HF-PEBF (3.96 CFU/g) at the end of the studied period. Counts for the low-fat sausages (LF-PEBF) showed no significant changes during storage (3.14 CFU/g). There are few studies of preservation in cooked sausages using flours as fat replacers, and the formulations are so different that it is not easy to compare results. Despite this, it is worth noting that similar results were observed in other meat products. So Bologna-type sausages with 2% citrus fiber reached 5 CFU/g for aerobic bacteria after 15 days of storage [Punica granatum and Citrus spp. ranged from 3.8–5.19 log CFU/g (depending on the formulation) after 2 weeks of storage [Initially, the counts of mesophilic aerobic microorganisms were 2.4–3 log CFU/g for all lots A. Simila storage , and por storage . Cerón-G storage  observedp < 0.05). Meat product quality could be affected by the inclusion of flour -and its starch content- due to the presence and/or increment of mold and yeasts [Mold and yeast counts were evaluated due to the inclusion of different quantities of flour in the formulations. A similar pattern to that described previously for total mesophilic aerobic counts was observed B, with Ld yeasts , given td yeasts .Enterobacteria and Staphylococcus aureus, no counts were obtained during chilled storage in any lots. Heat treatment, as well as the addition of nitrites into the formulation, inhibits the growth of these microorganisms due to a halt in active transport, oxygen absorption, and oxidative phosphorylation in bacteria such as Escherichia coli, S. aureus, and Clostridium botulinum [Regarding otulinum . The higp < 0.05), occasionally differing during storage by less than 0.5 points, which can be considered as negligible. Moreover, a slight increase in attribute scores with respect to the Control was observed in the high-fat sausages during this period; as in HF-PUBF for odor, taste, and acceptability and HF-WBF for odor (p < 0.05). The main difference found among the high-fat sausages was odor, increasing the score for this attribute during storage. No off-flavors were detected in any of the sausages during the studied period. Regarding the low-fat and flour-enriched sausages, sensory attributes were lower. Low-fat sausages were described to have an uncharacteristic smell and taste, a less pleasant texture, and therefore, the overall acceptability was lower. Regarding texture acceptability, the lower values may be due to the high hardness and low chewiness values in this sample that were obtained by mechanical methods, as previously described in p < 0.05), which may indicate that the judges became more accustomed to the product with time; in fact, judges perceived these sausages as a different product. This result is in accordance with Arildsen Jakobsen et al. [The incorporation of green banana flours hardly changed the sensory attributes of high-fat sausages . Controln et al. , who notn et al. . As the n et al. , or the n et al. , leads tThe use of banana flours  is an ideal alternative to wheat flour to produce Frankfurt-type sausages since they have physicochemical and composition characteristics that do not differ substantially among them, in their preservation or their sensory properties. This would allow new applications at an industrial level, favoring the creation of added value products from this underused material in certain countries, with good nutritional properties and low allergenicity, gluten-free. These new Frankfurt-type products could be labeled as a “source of fiber,” except for HF-PEBF, which could bear the “high fiber” claim.The low-fat and high-fiber sausages are different products in composition and physicochemical properties. These new sausages present higher values for hardness and a darker color, however, they preserve microbial growth-bacteria and molds and yeasts; with sensory properties in the acceptance limit, yet increasing acceptability with time when sausage awareness increases, as observed in tests performed during storage. So low-fat sausages formulated with banana flours may be candidates for consumption, especially if they are introduced as a new product, which can be labeled as “high in fiber” according to nutritional claims.The results obtained in this research allow establishing the viability of using banana flours as a meat extender in Frankfurter-type sausages, conferring good nutritional and healthy properties.


Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system. Phase separation in biological membranes has significant functional implications. Liquid-ordered lipid domains (LODs) and liquid-disordered domains (LDDs) exhibit different permeabilities to small molecules and dissimilar mechanical properties ,2. Proteα, spontaneous monolayer curvature J0 is the primary determinant of domain size )0 values  of LODs


K′ and K″ parameters of the power law models  and the highest values of the instantaneous (J0) and retardation (J1) compliances . Replacing the spelt wheat flour with the other ancient wheat flours and quinoa flour increased the proportion of elastic properties and decreased values of the J0 and J1 of the pasta dough. Presence of the quinoa flour increased pasting temperature (from 81.4 up to 83.3 °C) and significantly influenced pasting viscosities of the spelt wheat pasta samples. This study indicates a potential for using mixtures of spelt, einkorn, and emmer wheat flours with quinoa flour in the production of innovative pasta dough and pasta products.The aim of this study was to investigate thermal and rheological properties of selected ancient grain flours and to evaluate rheological properties of mixtures thereof represented by pasta dough and dry pasta. Flours from spelt, einkorn, and emmer ancient wheat varieties were combined with quinoa flour. All these flour sources are considered healthy grains of high bioactive component content. Research results were compared to durum wheat flour or spelt wheat flour systems. Differential scanning calorimeter (DSC) and a rapid visco analyzer (RVA) were used to investigate the phase transition behavior of the flours and pasting characteristics of the flours and dried pasta. Angular frequency sweep experiments and creep and recovery tests of the pasta dough were performed. The main components modifying the pasta dough structure were starch and water. Moreover, the proportion of the individual flours influenced the rheological properties of the dough. The durum wheat dough was characterized by the lowest values of the  Over the last several years there has been a growing interest from scientists, farmers, and food producers in ancient cultivated grains: cereals, minor cereals, and pseudo-cereals . These gTriticum aestivum L. subsp. spelta (L.) Thell), einkorn (Triticum monococcum L. subsp. monococcum), and emmer (Triticum turgidum L. subsp. dicoccum). These wheat species represent the cultivated hulled wheat and they comprise a bridging species between the cultivated, i.e., bread wheat (Triticum aestivum L. subsp. aestivum) and durum wheat (Triticum turgidum L. subsp. durum (Desf.) van Slageren) and the wild wheats [Chenopodium quinoa Willd.) has attracted special attention from scientists for its composition, especially for being rich in proteins, lipids, fiber, minerals, vitamins, and phenolic compounds, with an extraordinary balance of essential amino acids [The ancient grains are widely represented by the ancient wheat species, including spelt  semolina contained the most moisture, while the quinoa (Qn) flour had the least . The moiO, TgM, and TgE of the tested flours were determined for the average equilibrium moisture content of the samples during storage. On the basis of the values of these parameters given in O and TgE with, respectively, wet gluten content  and starch content  of the flours, i.e., with amounts of the components responsible for creating the amorphous structure of flour particles. On the other hand, positive correlations of TgO and TgE with fat content of the flours  were found. The flour component mentioned above may have contributed to the loosening of the amorphous structure of flour particles. Therefore, it can be concluded that apart from the amount and structure of the amorphous region of the starch, the flour constituents coexisting with starch in a flour might control the Tg value [The glass transition temperature of a product with low water content determines the conditions for optimal storage, necessary to protect the product against changes at the diffusion level. The TgTg value .Og. The lowest value of this parameter was determined for the Qn flour, while the highest value was detected for the EkW flour. It can be assumed that the TOg values were also controlled by non-starch components of the flours, especially by protein, the amount of which was negatively correlated with the TOg . Lack of significant differences in both the TPg and the TEg within the tested samples  indicates similar structures and degrees of crystallinity of starches in the compared flours [Parameters of the thermodynamic gelatinization characteristics of the flours are summarized in d flours .g was shown in the DW semolina  were lower than the starch gelatinization temperatures determined in the respective flours , and fat content  of the flours tested were found. The EkW flour was characterized by the highest PT value (The pasting temperature (PT) indicates the minimum temperature required to gelatinize the starch . In a syPT value , which wThe DW flour and the ancient wheat flours were notable for both the maximum viscosity in the heating phase and the minimum viscosity in their pasting characteristics a. BrandoThe DW flour suspension developed the highest viscosity in the heating phase (PV), while that of the EmW flour developed the lowest viscosity . The PV The extent of lowering of the viscosity of the systems after reaching the PV, calculated as BD × 100/PV, was relatively the largest in the case of the EmW flour (approx. 42%), lower in the SW flour (approx. 39%), followed by the EkW (approx. 31%), and DW (approx. 28%) flours, and the lowest was in the case of the Qn flour (approx. 7%). These data indicate a relatively high sensitivity of the pastes of the flours produced from the ancient wheat varieties to high temperature and mechanical treatments. The DW flour system reached the highest FV among the tested flours, while the Qn paste turned out to be the least viscous . The reaThe FV values of the flour systems under study exceeded the MV values two and a half times or approximately twice for the EmW flour or EkW, DW, and SW flours, respectively, while the FV value of the Qn paste was almost equal to the MV value. These observations lead to the conclusion that starch contained in the EmW flour was the most susceptible to short-term retrogradation, while this phenomenon was limited for the Qn flour. In the latter case, it could also result from the relatively low starch content of this flour . The setJ0) is related to the energy of elastic stretching of the material network bonds due to applied stress; the compliance disappears when this stress is removed. The retardation (viscoelastic) compliance (J1) is, in turn, associated with breaking or transformation of the inter-particle bonds [Oscillatory measurements are used to characterize viscoelastic properties of a material without destruction of its structure. In the case of pasta dough, its viscoelastic properties can be described by means of a linear combination of viscous and elastic properties at a properly adjusted amplitude of deformation. Another way used to characterize the viscoelastic properties of the material is to measure the creep and recovery phenomenon that is associated with a reorientation of the bonds in the material . In the le bonds .G′ and G″ moduli were found for the DWD  were obtained  and, on the other hand, by lower values of the G″ and K″ as compared to the SWD  as compared to this parameter determined for the SWD. In this particular case, the increase in the λret demonstrated extended creep straining. The η0 of the pasta doughs under study was influenced by the recipe composition: the mixed samples were distinguished by significantly higher values of this parameter than the SWD and DWD  were characterized, on the one hand, by higher values of the  the SWD . Moreovecoveries . The crecoveries . This ag and DWD . These rG′, J0, and J1 were strongly statistically significantly correlated with moisture content  and with starch content  of the flours. These findings indicate that the differences in the values of the discussed rheological parameters were mainly due to the different way the starch of the particular flour constituting the pasta dough component was binding water. Moreover, statistically significant positive correlations of protein content of the flours with the values of K′ and K″ parameters of the power law models  were also found. According to the literature [K′ and lower J0 and J1 values , while there were no relationships of both the G″ and the η0 with moisture and starch contents. These observations suggest that the interactions among the individual dough components are important for determining the rheological properties of the dough. Similarly, the differences in the G′ and the η0 values were due to the different chemical compositions, mainly different fat content of the flours constituting the dough components.The results of correlation analysis showed that values of the terature ,52,53,541 values , as well1 values  comparinp = 0.013, and r = 0.963, p = 0.037, respectively) and negatively correlated with protein content  of the dry pasta [The RVA pasting curves of dry pasta samples are presented in ry pasta . These ory pasta  on the rOn the basis of the pasting characteristics of the pasta samples, it can be assumed that the diversification of the qualitative and quantitative composition of the pasta investigated here will be reflected in different culinary characteristics, especially for the optimal cooking time and degree of water absorption during cooking, while texture properties of the cooked pasta samples may be similar to each other.Durum wheat (DW) semolina , spelt wheat (SW) flour , einkorn wheat (EkW) flour, emmer wheat (EmW) flour, and quinoa (Qn) flour  were supplied by Makarony Polskie S.A. .Moisture content of the flour was determined by drying at 130 °C for 1.5 h in a forced air oven  according to the AOAC method 925.10 . Wet glu® differential scanning calorimeter . In order to determine the glass transition temperature, about 15 mg of flour was weighed into an aluminum pan. The temperature program consisted of successively heating samples to 150 °C for elimination of the enthalpy of relaxation of the amorphous samples, cooling to −60 °C, and then heating to 250 °C, at a heating and cooling rate of 10 °C/min. The obtained thermograms were analyzed with the Netzsch Proteus analysis software. The onset, middle, and end glass transition temperatures  were determined.Thermal analysis of flours was performed by using a DSC 204 F1 PhoenixOg), peak (TPg), and endset (TEg) gelatinization temperatures, as well as gelatinization enthalpy . The sample was then stored for 7 days at 4 °C and reheated under the same conditions as were used for the gelatinization test. The onset (TOr), peak (TPr), and endset (TEr) starch retrogradation temperatures as well as retrogradation enthalpy  were determined from the thermogram. Additionally, the degree of retrogradation  was calculated using the following formula: DR = ΔHr × 100/ΔHg. The results are presented as the averages of at least two test repetitions.In order to determine starch gelatinization and retrogradation parameters, 3.5 mg of flour (d.w.b.) and 10.5 mg of distilled water were placed in an aluminum pan. The sample was kept at room temperature for 24 h to equilibrate. Then, the sample was heated in the temperature range of 25–110 °C with a temperature change rate of 10 °C/min. The following parameters were determined from the thermogram: onset . A homogenous flour suspension  was made in a disposable aluminum canister and then loaded into the apparatus. A thirteen-minute pasting profile  was usedw/w), in DWD, SWD, M1D, and M2D, respectively.Four pasta dough formulations with various proportions of the individual flours in the total amount of flours were prepared, as follows: durum wheat dough (DWD) with 100% of the DW semolina; spelt wheat dough (SWD) with 100% of the SW flour; mix-1 dough (M1D) with 70% of the SW flour, 10% of the EkW flour, 10% of the EmW flour, and 10% of the Qn flour; mix-2 dough (M2D) with 50% of the SW flour, 20% of the EkW flour, 20% of the EmW flour, and 10% of the Qn flour. The criteria for selecting the pasta recipes were the following: product innovation, sensory, and processing characteristics of the raw materials used, as well as the costs of the latter . The pasG′) and loss (G″) moduli as a function of shear stress in the range of 1–10,000 Pa at a constant angular frequency of 1 rad/s. Mechanical spectra were determined in the linear viscoelasticity region at constant deformation amplitude (γ = 0.001) in the range of angular frequency of 0.1–100 rad/s. The experimental data were described by power law models [G′ is storage modulus (Pa), G″ is loss modulus (Pa), ω is angular frequency (rad/s), K′, K″ is experimental constants, n′, n″ is the slopes of the plots.Rheological properties of pasta dough were measured at 25 °C by means of MARS II rheometer , using a serrated parallel plate geometry . A disc with diameter of 35 mm was cut out from the dough sheet, placed into the measuring system, covered with paraffin oil, and left for 5 min in order to ensure stress relaxation and to achieve the desired sample temperature. The linear viscoelasticity range was determined by plotting the storage (w models :(1)G′=K′G″/G′) was calculated and its dependence on the angular frequency (ω) was plotted for each of the samples.Based on the values of the dynamic moduli, the tangent of phase shift angle , J0 is instantaneous compliance, J1 is retardation compliance (Pa−1), η0 is zero shear viscosity (Pa∙s), retλ is retardation time (s), t is real time of experiment (s), t1 is time after which stress was removed (s).Creep and recovery tests were performed at constant shear stress σrameters :(3)J(t)=2) was used as quality criterium for fitting the models to the experimental data. All the tests were performed in three replications.Parameters of the rheological models used were determined by a non-linear estimation module using the Levenberg–Marquardt algorithm. The determination coefficient  was prepared from the pasta dough  according to the procedure described in our previous work . BrieflyThree and a half grams (d.w.b.) of the ground pasta sample were mixed with 25 mL of distilled water in a disposable aluminum canister and then loaded into the apparatus . A programmed heating and cooling cycle was used: the suspension was intensively mixed for the first 10 s at a speed of 960 rpm, held at 50 °C for 50 s with simultaneous mixing at 160 rpm (this mixing speed was maintained until the end of the test), heated up to 95 °C in 6 min, held at 95 °C for 8 min, cooled down to 50 °C in 6 min, and then held at 50 °C for 10 min. The pasting parameters . The data were subjected to a one-way analysis of variance (ANOVA), and the means were compared using Duncan’s test at significance level of 0.05. The Pearson correlation coefficients between the parameters were calculated, and their significance was tested at significance level of 0.05.The results of the analyses showed significant differences in the chemical composition, thermal and pasting properties of the tested ancient grain flours, especially in comparison with the durum wheat semolina. The diversified rheological properties of the tested dough samples were mainly determined by the starch and water contents of the flour or flours and, in the case of four-component mixtures, by the proportion of the individual flours. The structures of the spelt wheat flour-based dough samples were stronger than that demonstrated by the semolina dough. Replacing the SW flour with the other ancient wheat flours and the Qn flour increased the share of elastic properties of the dough as well as decreased its deformability as measured by the creep and recovery test. These rheological properties of the multicomponent pasta dough were most likely due to the achievement of an appropriate  gliadin to glutenin ratio. This phenomenon enables us to assume that there is a potential in using mixtures of spelt, einkorn, and emmer wheat flours with quinoa flour in production of an innovative pasta dough and consequently a variety of pasta products. The outcomes of the RVA tests showed that substituting the SW flour with the flours from the other ancient wheat varieties and with the Qn flour increased the PT and PV of the flour systems. This phenomenon was most likely caused by higher PT and pasting viscosities of the EkW, EmW, and Qn flours as compared to the SW flour. The results of our study allow us to claim that the diversification of the qualitative and quantitative composition of the pasta under study will be reflected in different culinary characteristics, while texture properties of the cooked pasta samples may stay similar.


Nonribosomal peptide synthetases (NRPSs) are large, multi-modular enzyme templates for the biosynthesis of important peptide natural products. Modules are composed of a set of semi-autonomous domains that facilitate the individual reaction steps. Only little is known about the existence and relevance of a higher-order architecture in these mega-enzymes, for which contacts between non-neighboring domains in three-dimensional space would be characteristic. Similarly poorly understood is the structure of communication-mediating (COM) domains that facilitate NRPS subunit docking at the boundaries between epimerization and condensation domains. We investigated a COM domain pair in a minimal two module NRPS using genetically encoded photo-crosslinking moieties in the N-terminal acceptor COM domain. Crosslinks into the C-terminal donor COM domain of the partner module resulted in protein products with the expected migration behavior on SDS-PAGE gels corresponding to the added molecular weight of the proteins. Additionally, an unexpected apparent high-molecular weight crosslink product was revealed by mass spectrometric analysis to represent a T-form isomer with branched connectivity of the two polypeptide chains. Synthesis of the linear L-form and branched T-form isomers by click chemistry confirmed this designation. Our data revealed a surprising spatial proximity between the acceptor COM domain and the functionally unrelated small subdomain of the preceding adenylation domain. These findings provide an insight into three-dimensional domain arrangements in NRPSs in solution and suggest the described photo-crosslinking approach as a promising tool for the systematic investigation of their higher-order architecture. Photo-crosslink analysis reveals unexpected insights into the higher-order architecture of NRPS and the nature of crosslink isomers. NRPSs are subdivided into modules, with typically each module being responsible for the incorporation of one amino acid into the peptide sequence. Modules contain specialized domains that activate the amino acid under consumption of ATP, bind them covalently as thioester at a prosthetic 4′-phosphopantetheine group (Ppant) of the peptidyl carrier protein (PCP), and catalyze peptide bond formation with the residue bound to the adjacent module. Once the linear product is assembled, it is cleaved off from the enzyme template by one of several mechanisms, including hydrolysis or reductive cleavage of the thioester as well as, most prominently, cyclization with the N terminus or a side chain of the peptide sequence. The essential adenylation (A), PCP, condensation (C), and thioesterase (TE) domains that are responsible for these reactions can be further supplemented with optional domains that carry out chemical modification of the building blocks at various steps during the assembly, for example epimerization and N-methylation catalyzed by E and M domains.Nonribosomal peptide synthetases (NRPSs) are the protein templates for the biosynthesis of a huge variety of small bioactive peptide natural products, also referred to as nonribosomal peptides (NRPs).7 the modules are arranged in a co-linear fashion with the amino acid sequence of the NRP product. While this arrangement suggests an ordered and largely repetitive spatial organization of modules and domains, it still remains unclear if and how multi-modular NRPSs templates are organized in a three-dimensional, higher-order superstructure.8–12 Crystal structures were obtained of several didomain truncation constructs13–16 and of some mono- and di-modules with multi-domain arrangements.8,9,17–19 These structural insights have confirmed significant conformational domain mobility between neighboring domains. In particular, the PCP domain as a carrier of covalently bound substrates and intermediates travels large distances to reach the various catalytic centers of the domains it has to interact with. In solution FRET studies have correlated these motions with catalysis.20 A recent cryo electron microscopy study suggested that the domain and module arrangements in multi-domain constructs might be very flexible, on top of the mobility of individual domains.8 Crystal structures of a dimodular fragment revealed contacts between a formylation domain of module 1 and a C domain of module 2, the first example of domain–domain contacts of domains that are not neighboring in the primary sequence.9 Such contacts might be indicative for a superstructure, however, due to crystal packing effects their occurrence as crystallization artefacts cannot be ruled out. For these reasons, new and complementary techniques to probe the structure of multimodular and entire NRPS templates in solution are needed.In a typical linear NRPS,21,23–28 Multiple such interactions are required in NRPS templates with more than two subunits  with one module and the gramicidin S synthetase B (GrsB) with four modules . The intsubunits . Combina subunit . This cline NRPS . Initial17 The tag sequence had reasonable sequence homology to the cognate donor COM domain, suggesting it mimicked the binding of the latter in the COM complex. The tag sequence adopted an α helix and formed contacts not only with the acceptor COM domain, but also with an extended surface on the C domain grasping around the helix like a hand, suggesting that additional sequences outside the COM regions might be important for the interactions. Based on this structure, a helix-hand model was proposed for the interaction  module. This structure showed by serendipity the binding of an unrelated tag sequence at the protein's C terminus to the acceptor COM domain in the crystal lattice.eraction .17 We cond motif .22 Consi34 The benzophenone moiety of the unnatural amino acid p-benzoyl-phenylalanine (BzF) is a widely used photo-crosslinker that can be repeatedly activated with light of approx. 350–365 nm.35 The formed diradical is short-lived and typically inserts into C–H bonds of side chains and the peptide backbone in a distance range of 3.1 Å,36 although larger labeling radii are possible due to rotations and flexibility at the BzF side chain and the surrounding environment.35,37 Methionine side chains have been observed as preferred crosslink partners.38Genetically encoded photo-crosslinking amino acids have enabled the probing of protein–protein interaction interfaces in a position-dependent manner.BzF39 in the acceptor COM domain of TycB1 in the dimodular GrsA–TycB1 system  mapping of the crosslinks. We performed a positional scan with 1 system . We repod-Phe-Pro dipeptide.22,40 The sequence alignment in 21 The donor COM domains encompass residues 1072–1098 of GrsA (1098 aa) and residues 1062–1088 of TycA (1088 aa). The acceptor COM domain of GrsB1/TycB1 is defined from residue 2 to 16 (the starting methionine is removed), however, the helix-hand model proposes that residues up to approx. position 83 are involved in the non-continuous hand part . The proteins were produced by recombinant production in E. coli and carried a C-terminal His6 tag to facilitate purification by Ni-NTA chromatography. GrsA additionally contained an N-terminal SBP tag (streptavidin binding peptide).The first two modules GrsA/TycA and GrsB1/TycB1 from the gramicidin S and tyrocidine NRPSs are functionally interchangeable in forming a and part .17,22 InKd = (5.0 ± 0.9) μM for the interaction between GrsA and TycB1 using microscale thermophoresis (MST) (We determined a dissociation constant is (MST) .BzF and p-azidophenylalanine (AzF) by the genetic code expansion technology that relies on suppression of an amber stop codon through co-expression of an engineered pair of aminoacyl synthetase and tRNA in the presence of the unnatural amino acid.39,41 The idea of our photo-crosslinking approach is to identify residues involved in the interaction interface of the two proteins. A participating position would be revealed by either obtaining a crosslink with the photo-crosslinking amino acid incorporated at the respective position or by a position in the partner protein that is identified as the targeted residue of a crosslink. Mapping these positions on known or postulated structures allows the testing and refinement of structural models and possibly the proposal of new constraints of their architecture.We employed incorporation of BzF into the first 13 positions (S2 to A14) of the acceptor module TycB1. These residues represent the “thumb” and the first β sheet of the “palm”-motif in the helix-hand model  protein was UV-irradiated either in the absence or presence of the GrsA partner protein and then analyzed by SDS-PAGE and immunoblotting against the SBP-tag on GrsA  and TycB1 (125.2 kDa) proteins became visible. We refer to these as low (l), middle (m) and high (h) bands . The lowControl experiments with gradually truncated COM domains on GrsA or TycB1 confirmed that the appearance of the GrsA-dependent high and middle bands was dependent on the intact COM regions and became weaker with their gradual deletion Fig. S5.BzF in the donor COM domain.22Since the migration behavior of these bands was difficult to determine precisely on our standard acrylamide Tris–glycine gels (6%) with a standard molecular weight marker (highest marker band at 200 kDa) as shown in BzF position . In one case, the fragmentation data quality allowed us to pin down the crosslink site to S1096 . The middle and high bands of a photo-crosslink experiment using GrsA and TycB1(S5BzF) were excised from the SDS gel, digested with trypsin and analyzed by LC-MS/MS. In the middle band digest, at least two chromatographically distinct isobaric peptides with to S1096  and S6A†rosslink .496TYMI500 of GrsA was recovered with M499 as the crosslink site . Indeed, the bands representing the L- and T-form crosslinks were observed in all native and non-native combinations, however, with varying relative intensities  were exclusively found in the middle bands and the terminus-internal crosslinks (T-form) exclusively in the high bands, the heterotrimer model of the first hypothesis appeared very unlikely 47,48 and p-azidophenylalanine (AzF)41 at interior  and terminal (F1090) positions in GrsA, as well as at terminal positions  in TycB1 ,in TycB1 . To geneLC-MS/MS  and S10†LC-MS/MS .BzF and AzF were incorporated at position Y498 of GrsA right next to M499 that was identified by MS-mapping. Indeed, following incubation of GrsA(Y498AzF) with TycB1 and UV-irradiation we mapped the crosslink to peptide S2VFSK6 of TycB1 , and in particular of M499. This latter region is located in the small and mobile Asequence , suggestC domain of GrsA and the acceptor COM domain of TycB1 we attempted to conclude on the most likely underlying conformation of the GrsA–TycB1 complex. Notably, this endeavor is complicated by the fact that neither structures of the donor COM domain or a native COM domain complex nor of a PCP-E-C sequence of domains are available. The E domain of GrsA is an additional binding site for the PCP that is not represented in known structures of entire NRPS modules. We reasoned that the AC subdomain of GrsA will be partially dragged on the backside of the PCP to the catalytic domains, as observed in several crystal structures.17,18 Next to possible open structures with the PCP not being in functional domain contact, the three expected positions the PCP can adopt are those in contact with the catalytic centers of the A and E domains of GrsA and the C domain of TycB1. We term the respective conformations as transfer, epimerization and donor condensation conformations 16. To project possible localizations of the AC domain relative to the PCP-E ensemble  with GrsA in three different chemical states. 22 These results are indeed consistent with our conclusion that the epimerization conformation of GrsA is likely the relevant conformation for the T-form crosslink (see above), because in a previous investigation of domain conformations in solution we found that the formation of the aminoacyl-thioester favors back-binding of the PCP to the A domain to adopt a post-transfer conformation.20,53 Accordingly, upon addition of substrates, an equilibrium of possible conformations in GrsA should be shifted in disfavor of the epimerization conformation, in agreement with the observed decrease of T-form crosslink efficiency. Interestingly, these findings also suggest that photo-crosslinkers might be useful tools to address conformational dynamics in NRPSs.Since NRPS conformations can be shifted between different equilibria depending on the catalytic state of the protein,43,54 or in branched intermediates of intein-mediated protein splicing pathways.55,56 We suspect the lengths of the polypeptide chains originating from the branch point as well as the pore size of the polyacrylamide gels to be the decisive parameters for whether or not such isomers exhibit sharply differing migration behaviors in gel electrophoresis.In conclusion, we could show that a T-shaped crosslink of two polypeptide chains can have a completely different migration behavior on an SDS gel than its isomer crosslinked in L-form. To our knowledge, this is the first systematic study to investigate and directly prove such kinds of protein backbone isomers. Our insights on L- and T-form crosslinks should also be applicable to other types of branched polypeptide chains, such as in proteins posttranslationally modified with ubiquitin-like proteinsC domain of the NRPS template can be localized in spatial proximity to the interaction mediating COM domain interface of two subunits. To our knowledge, this is the first non-neighboring domain contact in 3-D space unraveled for NRPSs in solution. Our results suggest a rational approach to investigate the three dimensional packing of domains in multimodular NRPS on the molecular level by photo-crosslinking to unravel their higher-order architecture, which is mostly uncharted territory.By photo-crosslinking and peptide mapping we have shown that a functionally unrelated and in primary sequence non-neighboring Apara-l-Benzoyl-phenylalanine (BzF) and para-azido-l-phenylalanine (AzF) were from Bachem, propargyl-l-tyrosine (PrY) was from Iris Biotech. Solvents for HPLC were from VWR. PerfectoPro Ni-NTA agarose was from 5 PRIME and streptactin sepharose as well as desthiobiotin were from IBA. Antibodies for western blot analysis were from Roche (anti-His), and secondary antibodies were from GE Healthcare. Synthetic oligonucleotides were from Biolegio.Restriction enzymes were from Thermo Scientific, buffer reagents, antibiotics and media components were from Carl Roth. 22 Unnatural amino acids  were incorporated using nonsense suppression. For BzF and AzF the corresponding pEVOL plasmids and for PrY the plasmid pUltra-CNF were introduced into E. coli expression strains.39,41,48,57 Each Uaa was added in a final concentration of 1 mM to the growth medium. All assays of the proteins were performed in NRPS buffer  unless differently specified. For storage at −80 °C, proteins were flash–frozen in liquid nitrogen in presence of 10% glycerol (v/v).Recombinant proteins were expressed and purified using Ni-NTA or streptavidin affinity chromatography as previously reported.Fhot  to Fcold . For evaluation, the change in fluorescence was plotted against the decadic logarithmic concentration of the interaction partner in the dilution series. The Kd value was calculated from the data of the MST curves as sigmoidal fit using the software NT Analysis .MST measurements were performed with the Microscale Thermophoresis Monolith NT.115 . GrsA was fluorescently labeled using the Labeling Kit RED-NHS . The concentration of fluorescently labeled GrsA was 1 nM and TycB1 was added in a dilution series in NRPS buffer with 0.05% (v/v) Tween-20 as indicated. The intensity of the laser excitation (LED power) was adjusted to give fluorescence intensity values between 400 and 1200 counts. The intensity of the MST excitation (MST-Power) varied between 20 and 40%. The binding was quantified from the ratio of λ = 366 nm, 8 W, 1.5 cm distance to the UV lamp) for 45 min. All samples were analyzed by SDS-PAGE and visualized by Coomassie staining and western blot.Proteins (each 5 μM) were pre-incubated at 25 °C for 45 min and then irradiated with UV light  were incubated in the presence of 1 mM TCEP, 100 μM TBTA and 500 μM CuSO−1 in 2% eluent B . At the same time, a solution of sodium formate clusters in 50% 2-propanol was injected to enable post-run calibration of MS and MS/MS spectra. After 10 min of washing at 2% B, a 50 min gradient  was applied for the separation on a C18 nano column . MS settings: capillary voltage 1.200 V, mass range: m/z 150-2200. MS survey scans were performed with a cycle time of 3 s. After each survey scan, the 10 to 20 most abundant precursor ions with z > 1 were selected for fragmentation using collision-induced dissociation. MS/MS summation time was adjusted depending on the precursor intensity. The applied precursor isolation window and the collision energy were chosen depending on the precursor m/z and charge.SDS-PAGE bands of crosslink products were excised, destained, and proteins were reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, and digested with 400 ng trypsin at 37 °C for 2 h in the presence of ProteaseMax  according to the manufacturer instructions. The acidified supernatant (addition of formic acid to achieve pH 2–3) was then analyzed on an LC-MS consisting of an UltiMate™ 3000 RS LC nano system  connected to a maXis II UHR-TOF mass spectrometer with a CaptiveSpray nano-ESI source  equipped with a nanoBooster device. For all proteins, 10 μL of the digest solution were injected. The solution was loaded on a C18 trapping column  at a flow rate of 20 μL min44DataAnalysis 4.4  was used for chromatogram processing and ProteinScape 4.0.3  was used to search our in-house database and for further analysis of MSMS data. Crosslink peptides were identified using StavroX 3.6.6 .There are no conflicts to declare.SC-011-D0SC01969K-s001


Interactions involving Epstein–Barr virus (EBV) LMP2A and Nedd4 family E3 ubiquitin–protein ligases promote the ubiquitination of LMP2A-associated proteins, which results in the perturbation of normal B-cell signaling. Here, we solved the solution structure of the WW2 domain of hAIP4 and investigated the binding mode involving the N-terminal domain of LMP2A and the WW2 domain. The WW2 domain presented a conserved WW domain scaffold with a three-stranded anti-parallel β-sheet and bound two PY motifs via different binding mechanisms. Our NMR titration and ITC data demonstrated that the PY motifs of LMP2A can recognize and interact weakly with the XP groove of the WW2 domain (residues located around the third β-strand), and then residues between two PY motifs optimize the binding by interacting with the loop 1 region of the WW2 domain. In particular, the residue Val15 in the hairpin loop region between β1 and β2 of the WW2 domain exhibited unique changes depending on the terminal residues of the PY motif. This result suggested that the hairpin loop is responsible for additional interactions outside the XP groove, and this hypothesis was confirmed in a deuterium exchange experiment. These weak but wide interactions can stabilize the complex formed between the PY and WW domains. Epstein–Barr virus (EBV) is present in all populations, infecting > 95% of humans, and EBV infection persists asymptomatically during the life of the host . EBV is Atrophin 1 Interacting Protein 4 (AIP4)  is a HECT-type E3 ubiquitin–protein ligase and is a member of the neural precursor cell-expressed developmentally downregulated gene 4 (Nedd4) family . VariousWW domains are small protein modules  that contain two conserved Trp residues that play crucial roles in the domain structure and function ,31,32. WLMP2A PY motifs selectively bind to the WW domains of ubiquitin–protein (E3) ligases. LMP2A recruits Nedd4-like ubiquitin protein ligases through the PY motifs, leading to the ubiquitination of LMP2A and several LMP2A-associated B-cell tyrosine kinases, such as Lyn PTK. Ubiquitination of these proteins perturbs normal B-cell signaling by rapid degradation of LMP2A-associated proteins . Thus, LWe previously reported that residues between two PY motifs of LMP2A may be involved in binding to WW domains directly and/or indirectly . In addi61 to L96): the region between the two PY motifs excluding both PY motifs; N-PY (from E54 to L96): containing the first PY motif  and the linker region; C-PY (from E61 to P103), containing the second PY motif  and the linker region. The constructs were prepared using the pTWIN1 vector with N-terminal Ssp DnaB intein fusion, and cloning, expression, and purification were performed as described previously HSQC spectra of the [U-15N] WW2 domain titrated with various unlabeled LMP2A constructs were obtained at 303 K on a Bruker DRX 500 spectrometer. The WW2 domain and added constructs were dissolved in 50 mM sodium phosphate (pH 6.0), 100 mM sodium chloride, and 7% D2O. Unlabeled LMP2A constructs  were added to the 50 µM [U-15N] WW2 domain at various molar ratios. The spectra were processed and analyzed using NMRPipe/NMRDraw [1H and 15N) upon binding the LMP2A constructs were calculated using Δδave = [(Δδ2HN + Δδ2N/25)/2]1/2 where ΔδHN and ΔδN are the amide proton and nitrogen chemical shift differences, respectively ; u; u48]; uW domain , which mBased on our results, we propose a model of the interaction between human AIP4 and LMP2A . Human AMaintaining latency in infected cells and tumorigenesis by LMP2a may be related with the recruiting ability of E3 ligases that provoke the unwanted proteolysis of signal molecules such as protein tyrosine kinases. It is known that Syk is a main molecule in LMP2a signaling  and dramThis study provides structural insights into the interaction between two PY motifs of LMP2A NTD and WW domains of the E3 ubiquitin–protein ligase. The XP groove of the WW2 domain may provide a binding pocket for LMP2A NTD PY motifs, and this binding may be optimized by additional interactions between the linker region and WW2 domain. In this process, the hairpin loop region between strands β1 and β2 is likely to act as a sensor for detecting different chemical environments. Further studies of the interactions between PY motifs and WW domains, especially complex structure of two consecutive WW domains and LMP2A NTD, would contribute to understanding the mechanisms and role of the ubiquitination process of LMP2A, which will eventually enable us to elucidate the general mechanisms involving binding between PY motifs and WW domains. Recently, proteolysis targeting chimera (PROTAC) techniques have become popular to discover novel drugs, which overcome the traditional small chemical drug. It uses cellular E3 ligase systems to degrade target proteins through physically linking between E3 ligases and target proteins . Our stu