Km value of the phosphorylation is increased from 10 to 45 μM upon additional deletion of the C-terminal SARAH domain. On the other hand, deletion of the flexible loop (Δ177–197) that precedes the phosphorylation site/s (T202/S203) results in a reduction of the kcat value from about 40 to 7 min−1. Direct physical interaction between the isolated SARAH domain and Aurora A was revealed by SPR. These data demonstrate that the SARAH domain of RASSF1A is involved in the binding to Aurora A kinase. Structural modelling confirms that a novel complex is feasible between the SARAH domain and the kinase domain of Aurora A. In addition, a regulatory role of the loop in the catalytic phosphorylation reaction has been demonstrated both experimentally and by structural modelling.The tumour suppressor protein RASSF1A is phosphorylated by Aurora A kinase, thereby impairing its tumour suppressor function. Consequently, inhibiting the interaction between Aurora A and RASSF1A may be used for anti-tumour therapy. We used recombinant variants of RASSF1A to map the sites of interaction with Aurora A. The phosphorylation kinetics of three truncated RASSF1A variants has been analysed. Compared to the RASSF1A form lacking the 120 residue long N-terminal part, the  RASSF1A protein is involved in three important cellular processes: microtubule stability, mitosis and induction of apoptosis. Loss of function of RASSF1A leads to accelerated cell cycle progression and resistance to apoptopic signals, resulting in increased cell proliferation. Thus, the development of targeted drugs to restore RASSF1A function is a desirable goal. In order to achieve this, the phosphorylation status of RASSF1A, sensitively influencing its various functions should be controlled.The tumour suppressor gene Ras-association domain family 1 isoform A (RASSF1A) is frequently silenced in a wide range of cancers. The exact mechanism by which RASSF1A exerts its tumour suppressor effects has not been clarified5 or protein kinase A6. Phosphorylation of RASSF1A by Aurora A on Thr 202 and/or Ser 203 disrupts its association with the microtubule network, thereby allowing mitotic progression4. The Aurora A mediated phosphorylation also suspends the mitotic blockade caused by RASSF1A through interaction with the complex of Anaphase-Promoting Complex and Cell division cycle protein 20 (APC/CCdc20)5. Furthermore, the activated association of APC/CCdc20 ubiquitylates RASSF1A, priming it for degradation. Thus, Aurora A mediated phosphorylation of RASSF1A promotes mitotic progression by causing APC/CCdc20 activation and subsequent degradation of RASSF1A7.RASSF1A is known to be phosphorylated by certain cellular kinases, such as the mitotic kinase Aurora A11. However, inhibiting a multifunctional protein might cause disruption of its other essential physiological functions. For this reason, targeting of a particular protein-protein interaction, such as Aurora A kinase-RASSF1A enzyme-substrate interaction could be the solution. Although there are numerous structural and functional studies with Aurora A kinase15, information on the isolated RASSF1A and its functional complex with Aurora A is still lacking. Up to now, interaction of RASSF1A with Aurora A kinase has only been demonstrated qualitatively by pull-down experiments16.The mitotic Aurora A kinase is often targeted by specific inhibitors as potential drugs against cancerin vitro kinetic phosphorylation assay. We found that the isolated full-length RASSF1A exhibited an extremely strong tendency for aggregation. This may be due to the long unstructured N-terminal region  are probably entirely different.In this work, we aim to quantitatively characterise the enzyme-substrate interaction between the isolated Aurora A kinase domain and RASSF1A using an 2. Additional deletion of the SARAH domain yielded RBD, which was also examined in order to test the contribution of SARAH domain to the catalytic complex formation with Aurora A. Another aspect of our kinetic studies was to test the previously assumed role21 of the flexible loop (residues 177–197) preceding the phosphorylation site/s (T202/S203) in the functional complex formation with Aurora A. For this purpose, the loop-deleted variant of ΔN (i.e. ΔN-Δloop) was also expressed and studied. All these truncated variants are schematically illustrated in Fig. The ΔN fragment consists of the Ras-binding domain (RBD), where the phosphorylation site is located, and the attached C-terminal Sav/Rassf/Hpo (SARAH) domain, a domain often involved in protein-protein interactions23. To determine if the RASSF1A variants used in this study form dimers, we performed analytical size-exclusion chromatographic experiments. Since the dimerization state of proteins is dependent of their concentration, the ∆N and RBD constructs were diluted to a series of different concentrations. These samples were then injected to a Superose 6 column. The resulting chromatograms are shown in Fig. It is known that SARAH domain containing proteins can form homo- and heterodimers through this domainThe protein samples of ∆N and RBD are eluted in a single major peak, with some inhomogeneities detectable at higher molecular masses, indicating their propensities for easy aggregation. The SARAH domain construct was also eluted as a single peak. The chromatographic column was calibrated using a series of proteins of known molar mass. The calibration data was then used to determine the molecular masses corresponding to each peak Table . In the Thus, the size exclusion chromatographic experiment with RBD clearly indicates the existence of its monomeric form, at least within the investigated protein concentration range. For the ∆N variant, as well as for the isolated SARAH domain, the experiments indicate equilibrium between monomeric and dimeric states, with association/dissociation rates faster than the rate of their possible chromatographic separation. It is evident that the dimerization ability of ∆N is due to its SARAH domain.in vitro phosphorylation experiments were performed on deletion mutants of RASSF1A. A constant concentration of Aurora A was incubated with various concentrations (up to 20 μM) of each RASSF1A mutant in the presence of MgATP partially labelled with radioactive 32P on its γ-phosphate group. By measuring the intensity of radioactive labelling incorporated into the RASSF1A substrate, the amount of phosphorylated RASSF1A could be calculated.To study the functional interaction between Aurora A and RASSF1A, The data yielded by these experiments are illustrated on Fig. Km value of the reaction is significantly increased compared to ∆N, suggesting that the SARAH domain increases the affinity of RASSF1A to Aurora A. This finding suggests direct physical binding of the SARAH domain to the surface of Aurora A. Deletion of SARAH domain also increases kcat slightly. A possible explanation for this effect is that by stabilizing the E*S complex, the SARAH domain may also hinder its dissociation. This would also imply that – at least in the presence of the SARAH domain – dissociation may contribute to the rate limiting step of the reaction, i.e. the classical fast-equilibrium Michaelis-Menten mechanism may not entirely hold.For RBD (which lacks the SARAH domain), the Km, but kcat is much smaller for ∆N-∆loop. These data suggest that – in contrast with the SARAH domain – the phosphorylation loop has an important catalytic role, probably by providing flexibility required for the formation of the catalytically competent E*S complex conformation. The unchanged Km suggests that the loop itself does not contribute to the formation and stabilization of the physical interaction between RASSF1A and Aurora A.Comparison of the kinetic parameters determined for the ∆N and ∆N-∆loop variants shows no significant difference in To confirm this interpretation, we performed another phosphorylation experiment where Aurora A and RASSF1A ∆N were present in a constant concentration, and a synthetic peptide representing the loop (residues 177–197) was also added to the reaction mixture in varying amounts, up to a concentration of 1 mM. In this experiment, the presence of the peptide had no detectable effect on reaction velocity, proving its inability to disturb the formation of the E*S complex Fig. . TherefoThe kinetic data presented in the previous section show that substrate saturation cannot be reached  using lower concentrations of RASSF1A. Also, it seemed desirable to investigate if dimerization of ∆N has any effect on phosphorylation kinetics. Therefore, the experiments were extended to a higher  concentration range in the case of the ∆N (up to 160 μM) and RBD up to 45 μM) variants  experiments to test the binding of the separately expressed SARAH domain to the immobilised Aurora A kinase domain Fig. . Since aRBD Fig. . The defRBD Fig. , possiblRBD Fig.  comparedRBD Fig. .Figure 5To assist with the interpretation of the kinetic and binding results above, we have carried out modelling studies to determine a possible mode of molecular interaction between Aurora A and its substrate RASSF1A. Since our experiments were restricted to the truncated variants of RASSF1A, i.e. ∆N, ∆N-∆loop and RBD, our modelling was also carried out with these variants. Models were built for the RBD-Aurora and ∆N-Aurora complexes based on predicted structures for the RASSF1A variants and experimental structures of Aurora A.Km values obtained for the loop-deletion mutant (∆N-∆loop) and ∆N  is shown in Fig. 24. This is also confirmed by our size-exclusion chromatographic experiments with the isolated SARAH domain   is/are located next to a disordered loop on the RBD domain of RASSF1A cf. Figs . The X-rct) Fig. . We foun02/Ser203ion Fig. . Our strion Fig. , in fact30. Our gel-chromatographic experiments  of RASSF1A cf. Fig. . This isnts Fig.  clearly nts Fig. . In factlly Fig. . UnfortuΔN Table . This co25, unlike in our case where a SARAH domain is found to bind to a globular domain. Our structural modelling illustrates a possible way of this interaction . The unlabelled ATP was Sigma-Aldrich product. A synthetic peptide with the sequence corresponding to the deleted loop (cf. below) was synthesized by GenicBio Ltd. Company. All other chemicals used were commercially available, high purity products.The vector used for the expression of proteins was an altered version of the commercially available pET24c vector. The isotope-labelled γ-The following truncated variants of RASSF1A were used in the experiments: ∆N (residues 121–340), RBD (residues 121–290) and ∆N-∆loop . The SARAH domain (residues 291–340) was also expressed separately. Aurora A was also produced as a truncated construct, consisting of only its kinase domain (residues 107–403). The genes encoding the truncated protein mutants were created by using Polymerase Chain Reaction (PCR) to amplificate the appropriate regions of their respective wild-type genes. The primers contained restriction cleavage sites, so the PCR products could be cloned into a modified pET24c vector with a Maltose Binding Protein (MBP)-coding sequence upstream, and 6XHis tag coding sequence downstream to the multiple cloning sites. The RASSF1A loop deletion (∆N-Δloop) was introduced into the ∆N construct by whole plasmid PCR. The primers were designed to stick to sequences bordering the deletion region from both sides but lacking the target sequence . After the reaction finished, the mixture was treated by DpnI to get rid of the methylated parental plasmids, leaving the PCR products – carrying the deletion mutants – intact. The sequences of all mutants were confirmed by sequencing.E. coli Rosetta 2 cells using a modified pET24c vector. Cell cultures were grown on 37 °C, to an OD600 of 0.6–0.8 then cooled to 21 °C. Expression was induced by addition of 0.4 mM Isopropyl β-D-1-ThioGalactopyranoside (IPTG), and then continued overnight at 21 °C. Cells were harvested by centrifugation, resuspended in a buffer containing CompleteUltra protease inhibitor mix and lysed by ultrasound. The lysate was centrifuged, and the target protein was purified from the supernatant.All proteins were expressed with N-terminal MBP, and C-terminal 6XHis affinity tags, in All proteins were fused to an N-terminal MBP-tag, and were first purified using amylose affinity chromatography. The bound target proteins were eluted by 10 mM maltose. In the case of the Aurora A kinase domain, this step was followed by a nickel affinity chromatography , to separate the intact protein from its degradation products. This time 250 mM imidazole was used as eluent. All RASSF1A protein constructs have been identified by using SDS PAGE according to their molecular masses of 69, 63, 67 and 51 kDa for ΔN, RBD, ΔN-Δloop and the SARAH domain, respectively (including the MBP-tag).37. The solutions were aliquoted, frozen in liquid nitrogen and stored at −80 °C.All proteins were further purified using size exclusion chromatography on a Superose 6 column to separate the native product from the aggregate. The column buffer was 25 mM HEPES, 300 mM NaCl, 3 mM DTT , at a pH of 7.4. The purified proteins were concentrated, and their final concentrations determined by UV spectrophotometry using molar absorbances calculated on the basis of a previously published methodCleavage of the N-terminal MBP-tag by Tobacco Etch Virus (TEV)-protease was also tried in a single case of a truncated variant of RASSF1A followed by a cation exchange chromatography, however, the protein yield was dramatically diminished.Samples of different RASSF1A mutants were diluted in a buffer  to different concentrations. Of these dilutions, volumes of 100 μl were injected to a column filled with 30 ml Superose 6 gel filtration medium, and chromatographed using FPLC. Proteins were detected by measuring the absorbance of the eluate at 280 nm.2 and 2 mM DTT. The reaction was started by addition of 0.4 mM ATP, partly labelled with 32P isotope on its γ-phosphate. To determine the initial velocity of phosphorylation, reaction mixtures were incubated for 2 minutes at 25 °C, and then a sample was pipetted into a reducing SDS sample buffer, terminating the reaction. The samples were boiled, and then excess ATP was separated from the phosphorylated RASSF1A by SDS PAGE.To examine the initial velocities of phosphorylation of the different RASSF1A variants by Aurora A, reaction mixtures were prepared in a pH 7.4 HEPES buffer, with Aurora A kept at a constant concentration of 40 nM and the concentration of RASSF1A varied. The mixtures also contained 100 mM NaCl, 5 mM MgCl32P incorporation was visualized by exposing the gels to a GE Healthcare StoragePhosphor screen, then scanning the screen by a Typhoon TRIO+ scanner. The resulting image showed bands corresponding to the phosphorylated proteins, with their density related to the amount of phosphate incorporated during enzyme reaction. The densities were quantified using densitometry, and then converted to molar concentration using an appropriate calibration standard of known amounts of fully-phosphorylated RASSF1A. From these concentrations v0 values for each reaction could be determined – the samples were taken at the initial phase of the reactions, confirmed by the amount of substrate converted.v0 and [S]0 values were plotted and fit using the software Sigma-plot (version 11.0) to determine the kinetic parameters of the enzyme reaction for each variant of RASSF1A.Using the Michaelis-Menten model of enzyme kinetics, SPR binding experiments were carried out to test the binding of the isolated SARAH domain and the ΔN and RBD variants to the immobilised kinase domain of Aurora A using a Biacore X equipment. Aurora A kinase domain was immobilised as a ligand to a GE Healthcare CM5 chip. The different RASSF1A variants were used as analytes in the mobile phase. The signal was automatically corrected by that of a reference cell without any immobilised proteins. All experiments were carried out in a HBS-EP buffer  at a temperature of 25 °C.38. Chain B of PDB entry 3ddc21, a structure of murine RASSF5, was found to be the best template with a sequence identity of 0.58 over the aligned part, with a coverage of 0.6. The estimated TM-score of the best model was 0.43. Five models were built; these mainly differed in the conformation of the 177–197 loop region. Upon visual inspection, the structure with the most plausible loop conformation was chosen for further modeling.A homology model for the ΔN RASSF1A was constructed using the I-TASSER pipeline39) to model the kinase-peptide complex. The Aurora A structure 4dee40 (containing an ADP molecule) was fitted onto chain A of 4ib0 using TM-align41 and the PSP20 peptide was copied over into the Aurora A structure. Torsion angles of a 14-residue segment of PSP20 (7–20) were used as dihedral restraints for the homologous 193–206 segment of RASSF1A (containing the phosphorylated site), and short (50 ps) vacuum molecular dynamics simulation was carried out with these restraints on our ΔN RASSF1A model. GROMACS 5.0.242 was used with the CHARMM27 forcefield with default parameters at 300 K. The purpose of this simulation was to force the 193–206 segment of ΔN RASSF1A to a structure identical to that of the 7–20 segment of PSP20 in 4ib0. After this occurred, the ΔN RASSF1A-Aurora A complex model was constructed by a least-squares superposition of the ΔN RASSF1A 193–206 segment to the 7–14 segment of the PSP20 peptide that was previously copied into the Aurora A structure. This initial complex structure was used for further simulations.The building of a complex of ΔN RASSF1A with the Aurora A kinase required modeling the interaction of the substrate binding site of Aurora A with the phosphorylated residue of RASSF1A. Because there is no Aurora A structure with a bound peptide substrate, we used the structure of cAMP-dependent protein kinase in complex with PSP20, a 20-residue phosphorylated peptide  after superposing the kinase structures, and the Aurora A subunit in all 10 model complexes was replaced by this 1mq4-ATP complex. The 10 Aurora A-ΔN RASSF1A complexes were then subjected to 50 ns molecular dynamics simulations at 300 K in vacuum using GROMACS 201642. The Gromos54a7 forcefield46 was used; electrostatic interactions were treated using the Particle Mesh Ewald method47 and the LINCS algorithm was used to constrain all bonds48. The last structure from each trajectory was then subjected to energy minimization.The complex generated in the previous step was constructed without a SARAH domain. To add a SARAH domain, we used Modeller


Bos taurus indicus) has potentially increased in economic impact. The objective was to investigate the factors associated with leptospirosis in cattle in the protected area. We investigated the seroprevalence of leptospirosis in cattle in Salakphra Wildlife Sanctuary, Thailand. Serum was collected to investigate the seroprevalence by agglutination test and their associated factors. From a total of 513 samples, antibodies against Leptospira were detected in 92.2% of samples. Within a total of 42 herds, the serovar with the highest prevalence was L interrogans serovar Tarassovi (92.9%). Most leptospirosis was found in medium-sized herds with the highest concentrations in cattle farms close to cities . Seroprevalence was associated with herd size, raising pattern in the dry and wet seasons, grazing distance, number of years that cattle were kept in the farm, the introduction of new cattle into the farm, and keeping some pets in the farm. The results of the study suggest that keeping cattle in larger herds, raising pattern and distance, keeping period, and introducing new cattle and having pets impart potential risk of increasing leptospirosis exposure. These results indicate that cattle are important hosts of Leptospira in Thailand and may act as sentinels of Leptospira infection for wildlife and people in the protected areas.Leptospirosis found in cattle ( Bos taurus indicus)  is a risk factor for human leptospirosis [Leptospirosis affects humans worldwide . Occupations involving exposure to domestic cattle  in Thailand and is surrounded by farmland. Indeed, 50% of this protected area is invaded by domestic animals. Chaiyarat and Srikosamatara [Salakphra Wildlife Sanctuary is located in the Western Forest Complex (WEFCOM)  (Elephas maximus), gaur (Bos gaurus), and sambar deer (Rusa unicolor) can be found [Salakphra Wildlife Sanctuary is located in Kanchanaburi province  . The arebe found .Animal treatment protocols were approved by the Ethics Committee of Mahidol University, Thailand in 2017. The study was conducted on cattle herds totaling more than twelve thousand animals based on estimates by park rangers. The sample size for this study was estimated by WinEpiscope 2.0  at 10% of the total individuals in each cattle herd with 5% allowable error. At least 254 of the animals should be included in the study to achieve a 95% confidence interval. In this study, a total of 513 domestic cattle were randomly selected.All 42 of the selected herds located in the 5 km boundary of Salakphra Wildlife Sanctuary were visited in this research, resulting in a 100% response rate for participation in the study. The average herd size was 62±32 animals. The primary purpose of raising domestic cattle was for sale to the local slaughterhouses (95.2%). All herds had feeding supplements such as hay and mineral blocks. For healthcare management, 97.6% of herds were vaccinated with foot-and-mouth disease vaccine and 85.7% used ivermectin for deworming routinely. Sixty-two percent of the herds had a sign of sickness such as lameness, wounds, and hoof problems.Leptospira interrogans antibodies by means of the Microscopic Agglutination Test (MAT) as previously described [Leptospira interrogans were tested: L. interrogans serovar Tarassovi, Ranarum, Hebdomadis, Ballum, Bratislava, Sejroe, Autumnalis, Pyrogenes, Bataviae, Sarmin, Canicola, Djasiman, Icterohaemorrhagica, Pomona, Cynopteri, Mini, Javanica, Louisiana, Shermani, and Patoc I , rarefaction, and similarity index (Kulczynski comparison) were calculated by using BioDiversity Professional Version 2 [The diversity index  was used for mapping the relationships between environmental factors and seroprevalence in domestic cattle . InformaMaxomys surifer), is as followsH′ = Diversity index of Shanon-Weiner. ip = The proportions of the many capabilities of the seroprevalence i compared to all of the seroprevalence (N) = in/N when i = 1, 2, 3,…, S. S = number of seroprevalence in cattleThe Shannon–Wiener diversity index , used top < 0.05. Statistical analyses were performed using SPSS. The relationships between the herd variables and the seroprevalence were analyzed via the correlative coherence analysis (CCA) with PC-ORD 5.10 [A chi-squared test was employed to test differences between leptospirosis and the associated environmental factors. Differences were determined to be significant based on the ORD 5.10 .L interrogans serovar Tarassovi (92.9%). The highest titer observed was 1:800  had antibodies against at least one of the 20 Leptospira serovars, which were used at a titer equal to or above 1:50. Overall, a total of 42 herds, the serovar with the highest herd seroprevalence was as 1:800 . The potas 1:800 .χ2 = 44.56, df = 6, p < 0.001), the presence of leptospirosis was not different among districts . There was no difference in raising patterns of domestic cattle between dry and wet seasons. In both seasons, a few herds were kept in the village and most of them were moved to the forest for free grazing. The raising distances from farms in Salakphra Wildlife Sanctuary were different. Most herds were raised in distances of more than 5 km . The number of years that the domestic cattle were kept in the farm was different. Most of them were kept longer than 10 years . Introduction of new animals into the herd  and pets on the farms  were different. However, the category raising the same route with other herds, sharing the feeding ground with other herds, and abortion histories were not different   was higher than red spiny rat  with χ2 = 173.4 . The similarity between cattle and red spiny rat were 62.2% .The relationship between the herd seroprevalence  and the Serovars Canicola, Mini, Pomona, Pyrogenes, and Sarmin were correlated with the number of years that domestic cattle were kept in the herds, the distance of raising from the households, and the location of herds. While serovars Autumnalis, Ballum, Cynopteri, Djasiman, Icterohaemorrhagica, and Javanica were associated with the introduction of new animals into the herds and the abortion history of the herds .The seroprevalence found in this study was higher than a recent survey of cattle (17%) of nearby protected areas in Kanchanaburi province  as well Leptospira spp. The real prevalence  was probably the opposite. It could be suggested that the diseases were not transmitted only by rodents and domestic cattle but could be an incidental infection caused by strains carried by other domestic or free-living animals [The seroprevalence in this study did not match previous studies in domestic cattle in Thailand by Laoluekiat and Jongpipatvanitch , who fou animals . However animals  and Indi animals . Leptosp animals .Management factors have potentially influenced the transmission between domestic cattle and red spiny rats. Cattle raised in herds for over ten years faced a higher risk-the longer the infected animals remained in contact with the remainder of the herd, the greater the number of seropositive animals . For a mFinally, our study supports the idea that keeping domestic cattle within an area with a high risk of contact with other animals has a potential risk of leptospirosis transmission among domestic cattle . The potL. tarassovi, L. ranarum, and L. hebdomadis were most commonly found in the area. These species are also found in a domestic Asian elephant camp nearby the Salakphra Wildlife Sanctuary. Other environmental factors that were associated with leptospirosis in the Salakphra Wildlife Sanctuary were domestic cattle raising patterns, raising distance, keeping periods, the introduction of new animals into the herds, other pets kept on the farms, and distance from villages. We suggested that domestic animals should be fenced away from sharing habitat with other wildlife to reduce the potential transmission of Leptospira in the area and elsewhere.Most domesticated cattle in the Salakphra Wildlife Sanctuary that were kept in medium-sized herds had higher seroprevalence than other herd sizes.


Spatially resolved information about material deformation upon loading is critical to evaluating mechanical properties of materials, and to understanding mechano-response of live systems. Existing techniques may access local properties of materials at nanoscale, but not at locations away from the force-loading positions. Moreover, interpretation of the local measurement relies on correct modeling, the validation of which is not straightforward. Here we demonstrate an approach to evaluating non-local material deformation based on the integration of nanodiamond orientation sensing and atomic force microscopy nanoindentation. This approach features a 5 nm precision in the loading direction and a sub-hundred nanometer lateral resolution, high enough to disclose the surface/interface effects in the material deformation. The non-local deformation profile can validate the models needed for mechanical property determination. The non-local nanometer-precision sensing of deformation facilitates studying mechanical response of complex material systems ranging from impact transfer in nanocomposites to mechano-response of live systems. Spatially resolved information about material deformation upon loading is critical to evaluate mechanical properties. Here the authors demonstrate integration of nanodiamond orientation sensing and atomic force microscopy nanoindentation as an approach to evaluate non-local material deformation on the nanoscale. Nanoindentation8, a method that gives the loading depth profile of materials, is commonly used to investigate the mechanical response upon loading10. Although local deformation at the contact region can be obtained by well-established methods14, the resolution of the measurement remains unsatisfactory. More importantly, the measured local deformation results from a summation of sample deformation and cantilever deflection. It largely depends on the conditions of the indentation, such as the type of indenter, the indentation speed, the type of liquid employed, the humidity, etc.16. If soft matter or biological systems are studied, the viscosity of the samples also plays a role. Using only local deformation profiles is usually inadequate to determine the material mechanical properties. On the other hand, the established methods miss the information about the non-local response of the material to the local loading; such information is essential to understanding the deformation and impact transfer in heterogeneous structures, and how live systems respond to mechano-stimuli. Up to date, the only deformation measurement method that can extract non-local information is based on optical imaging22. The precision and the resolution of the optical measurement, however, is limited by the optical wavelength; or even with the super-resolution techniques based on single-molecule fluorescence, they are still limited to tens of nanometers. In typical optical measurement of indentation-induced deformation, indenters of large tip size (a few micrometers) are usually used to generate deformation large enough to be measurable (>a few micrometers)22. Such techniques lack the sensitivity required to probe important deformation phenomena, such as the effects of surface tension at the interfaces between soft materials and liquids. This is particularly relevant to the mechanical property measurement of soft matters, such as single cells, when accurate determination of the stiffness cannot be done without knowledge of the elastocapillary effect23. Therefore, it is highly desirable to develop a new method that can have both non-local access of deformation and nanometer precision. Such a tool would not only enable the investigation of impact transfer at the nanoscale in heterostructured materials with multiple phases and interfaces, but also facilitate the study of fine deformation characteristics of soft materials under weak external perturbation.Deformation measurements of heterogeneous nanomaterials26. Diamond sensing uses nitrogen-vacancy (NV) center27 spins as the sensor, which can work under ambient conditions32. NV center spin resonances are sensitive to small changes of the magnetic field, making them effective quantum sensors to many physical parameters via direct or indirect magnetometry measurements36. In particular, since the spin resonance frequencies are shifted mostly by the magnetic field component projected to the nitrogen-vacancy axis, vector magnetometry has been developed to determine the relative orientation between the magnetic field and the crystallographic direction of the diamond lattice39. The deformation is related to the orientation of the surface and hence the rotation of the nanodiamonds (NDs) that are situated on the surface. Based on this, with the combination of atomic force microscopy (AFM)-induced indentation, we propose and demonstrate a scheme of non-local deformation sensing using the rotation measurement of NDs on a surface.A new opportunity is diamond sensing40; this good agreement validates the method. We further apply this sensing method to measure deformation of gelatin microgel particles in water, using the multi-ND approach. We measure the effect of surface tension at the gelatin/water interface under nanoscale indentation. The load-responsive sensing protocols based on the ND rotation measurement provide an approach to studying spatially resolved non-local deformation, which features a precision <10 nm, bringing opportunities to study mechanical properties of nonuniform media and paving the way for investigating mechanical properties/mechano-stimuli-induced response of live systems in the future.We reconstruct the non-local surface topography using the three-dimensional rotation data of NDs docked on surfaces that are deformed by AFM indentation. We adopt two data-acquisition approaches—one with an ND fixed on a position and the AFM indentation position scans around the ND (single-ND approach); another with a fixed indentation position and multiple NDs locates at various positions on the surface (multi-ND approach). In the proof-of-the-concept demonstrations, we employ the single-ND approach and reconstruct the deformation of a polydimethylsiloxane (PDMS) film with a 5-nm precision in the loading direction and an in-plane spatial resolution ~200 nm (as determined by the AFM parameters—the scanning step size in the present experiments). The measured mechanical property of the PDMS film is well described by a linear elastic model that has been established in the literatureFigure 27 , S = 1) with a zero-field splitting D≈2.87 GHz between the mS = 0 and mS = ±1 states, which are quantized along the NV axis. In the presence of a weak magnetic field B (<100 Gauss), the frequencies of the transitions ψi is the angle between the NV axis (NVi) and the magnetic field B , with θr and ϕr being the polar and azimuthal angles, and the rotation angle χ  of the ND were deduced from the ODMR spectra before and after the rotation . The position of the ND is denoted as ρ = , written in the polar coordinates. The relation between the rotation of the ND and the rotation of the tangential plane of the surface at ρ, as illustrated in Fig. z-axis). The displacement of the surface at the position of the ND, denoted as z(ρ), was reconstructed by the least-square fitting of the rotation data, using the gradient field equation g(ρ) = ∇ρz(ρ)41, where the gradient is related to the rotated normal direction by n =  (for details see Methods). In reconstructing the deformation, we assumed that the attached NDs had no relative motion or rotation against the surface. This assumption is consistent with the fact that the NDs recovered their original orientation after the indentation was released  are shown in blue and green lines in Fig. B or B' with or without the indentation  of the ND from the AFM indentation positions, shown in the figure  was reconstructed from the rotation data of the ND as a function of the displacement from the AFM tip, as shown in Fig. χ . The simulated rotation angle χ of the ND and the deformation z, plot respectively in Fig. The rotation and deformation data are best described with linear elastic bilayer numerical simulations46 and Derjaguin–Muller–Toporov (DMT) model47, which are commonly used for studying adhesive materials, the two models generate similar fitting for a local deformation profile, though with different mechanical parameters , but the mechanical parameters of the material can only be determined when a correct model is employed, and the choice of the model relies on accurate knowledge of tip parameters and indentation conditions, the determination of which is not straightforward in many cases. As a result, one would not be able to validate the choice of contact models and consequently determine the mechanical properties of samples based on the local deformation profiles. For example, if one considers Johnson–Kendall–Roberts (JKR) modelThe second protocol utilized multiple NDs to map the deformation around the indentation imposed at one spot. We demonstrated this multi-ND sensing of deformation induced by a single nanoindentation on a gelatin particle in water Fig. . By measx–y plane and their lengths representing the rotation angles.We synthesized gelatin microparticles and dispersed the NDs on the surface in water (for details see Methods). Figures x–y plane and the NDs all rotated toward their respective indentation locations  around eight different indentation locations. In all cases, the rotation axes of all measured NDs were found approximately on the 23. The elastocapillary phenomenon has been disclosed as an important reflection of competition between the bulk elastic energy and surface energy23. However, the elastocapillary phenomenon only becomes significant when the scale of deformation is smaller than the elastocapillary length scale L = τ0/E (τ0 is the surface tension and E is the Young’s modulus). To have an observable elastocapillary effect, one either requires that the materials be very soft (with low E) and/or have high surface tension (large τ0), or is able to measure a small deformation to high precision .The high precision and the small AFM tip allowed us to study an important effect in deformation upon loading—the effect of surface tension. The surface tension has recently been identified as a key factor in affecting material deformation upon loading48 . Our results provide the first set of experimental data obtained in aqueous environment and demonstrate the role of surface tension in determining its mechanical properties, paving the way for future investigation of mechanical properties/mechano-stimuli-induced response of live systems.The identification of the surface tension in the gelatin measurement and the model validation are important for studying the mechanical properties of soft matters. In fact, recent studiesIn conclusion, we have developed a sensitive method to measure nonlocal nanoscale deformation by combining ND orientation sensing and AFM indentation. The technique features a high precision (~5 nm) in deformation along the loading direction, which is one or two orders of magnitude higher than that of the conventional fluorescence-based methods. The deformed surface profile obtained with high precision contains abundant information of the material mechanical properties, leading to the disclosure of heterostructures in PDMS films (due to surface oxidation), and to the first measurement of the elastocapillary effects at the gelatin/water interface.The two protocols, namely, the single-ND method and the multi-ND method, have complementary advantages and disadvantages. The single-ND method features a nanometer precision (~5 nm) in the loading direction, as determined by the rotation angle measurement precision, and sub-hundred nanometer lateral resolution, as determined by the AFM tip size or the scanning step size, whichever is larger. However, the deformation reconstruction based on the single-ND method relies on the assumption that the material is laterally homogeneous. For deformation reconstruction of laterally inhomogeneous materials, the multi-ND method is needed. The lateral resolution of the multi-ND method is determined by the optical resolution, or the density of the surface ND, whichever is larger. The sample preparation method in our current experiments results in a random dispersion of the NDs without much control on their density and locations. In the future, the surface functionalization of the NDs to achieve high dispersity, as well as control on the anchoring site, may be employed to improve the deformation reconstruction. Then the ultimate limit of the lateral resolution is the optical resolution.54 and high photostability of NDs and long spin coherence time of NV centers at room temperature27 make NDs attractive for a variety of bioapplications. Indeed, NDs have already been used for bioimaging/-tracking55 and drug delivery57. For potential biosensing applications, time resolution of 1 s is achievable with the current method  was thus spin coated on the PDMS film.We fabricated an Ω-shape microwave antenna on a glass slide and then spin coated PDMS on the top. The thickness of the PDMS was ≈50 μm. When the PDMS was degassed, we modified the PDMS surface with O−1 gelatin solution was added to the Span80 solution and the mixture was vigorously shaken for 20 s. After the shaking, the gelatin in toluene emulsion was stirred at 300 rpm and cooled under ambient conditions for 1 h. Then, the solidified particles were sonicated for 2 min. Finally, the particles were washed three times with 30 mL of acetone, and then three times with 30 mL of deionized water. The synthesized gelatin microspheres were spin coated onto a confocal dish with a microwave antenna (a 20-μm copper wire was bonded on the surface of a glass slide before a holder was glued on the glass slide to have a liquid chamber with microwave access). Then the ND solution was dispersed on gelatin microspheres using the drop-casting method.Gelatin (1.5 g) was dissolved in 10 mL of deionized water at 60 °C. The 0.15 g mLB and B'). In the gelatin indentation experiments, the magnetic field switching was implemented by a permanent magnet and an electric magnet .The setup was a homebuilt confocal microscope correlated with an atomic force microscope  is much larger than those of our samples (in the range of kPa to MPa). As a result, the tip deformation is negligible as compared with the sample deformation. With the contact point set as the snap-in point and the tip deformation taken as zero, we estimated the depth of indentations as the distance between the contact points and the force set points.−1 and deflection sensitivity: 42.28 nm V−1) was used, and the maximum indentation force was controlled at 150 nN. In the multi-ND method where gelatin was employed as the model sample, a PFQNM-LC-A AFM tip  was used, and the maximum indentation force was controlled at 15 nN.In the single-ND method where PDMS was selected as the model sample, a DNP10-A AFM tip  has about 500 NV centers. The ND solution was diluted in aqueous solution to 2 μg mL25The normalized ODMR spectra under the two known magnetic fields are written asb is the baseline, Ci represents the ODMR contrast of the NV centers along the ith crystallographic orientation, Δf is the linewidth (FWHM), and α,β,γ), see Supplementary Note 2–1 and Fig. D (slightly different among different NDs). Then, the rotation of the ND was determined by fitting the two ODMR spectra after the rotation with the rotation axis and angle , the contrasts, and the baselines as the fitting parameters. The Euler angles, the FWHM, and ΔD were fixed to be the values obtained by fitting the spectra before the rotation. For each ND, the contrasts and the baselines obtained by fitting the spectra before and after the rotation were approximately the same. For details of the fitting methods, see Supplementary Note where z(ρ) was reconstructed by the least-square fitting of the ND rotation data using the gradient field g(ρ)=∇ρz(ρ)41. The normal vector of the deformed surface is n = , which is related to the rotation of the attached ND as R is the rotation matrix with rotation axis  and rotation angle χ, and z-axis. Therefore, the gradient field was deduced from the rotation of the ND by The deformed surface 40. The Young’s modulus of the PDMS and the oxidation layer were chosen as 0.78 MPa and 9.4 MPa, respectively, the Poisson’s ratio was 0.33, and the thickness of the oxidation layer was 500 nm. The chosen parameters are consistent with the literature58 and with our AFM measurement. For details see Supplementary Note The indentation-caused deformation of the PDMS film was simulated with a half-infinite linear elastic model, with a harder coating layer under an axisymmetric loading48. The Young’s modulus was chosen as 6.5 kPa, the Poisson’s ratio was 0.5, and the surface tension of the water–gelatin interface was 6.5 mN m−1. The chosen parameters agree with the literature50. For details see Supplementary Note In the gelatin case, we used a half-infinite axisymmetric linear elastic model, including the effect of the surface tensionSupplementary InformationPeer Review File


This work provides a new theoretical approach based on group theory and graph theory to capture the intrinsic properties of crystalline materials upon deformation. The generation and motion of crystalline defects during plastic deformation are critical processes that determine the mechanical properties of a crystal. The types of defect generated are not only related to the symmetry of a crystal but also associated with the symmetry-breaking process during deformation. Proposed here is a new mathematical framework to capture the intrinsic coupling between crystal symmetry and deformation-induced symmetry breaking. Using a combination of group theory and graph theory, a general approach is demonstrated for the systematic determination of the types of crystalline defect induced by plastic deformation, through the construction of a crystal deformation group and a deformation pathway graph. The types of defect generated in the deformation of a face-centered cubic crystal are analyzed through the deformation pathway graph and compared with experimental observations. Thus, the DPG provides a new theoretical tool for tailoring material properties through defect engineering.2.D) using group theory and (ii) how to construct a DPG (HG) using graph theory, for a generic 2D example. Theoretically, a CDG is the coupling result of the crystal symmetry operations and an LID operation of a crystal. To visualize the group structure of the CDG, we construct its Cayley graph  how to construct a CDG . Furthermore, the internal structure of H is not the focus of our paper, as will be discussed below.Here, we first consider a square crystal lattice in 2D. The point group of a square lattice is 4H, d1 also corresponds to an operation that maps the square lattice onto itself. However, such a symmetry operation is associated with the translational symmetry of the lattice. Crystallographically equivalent deformations of d1 can be determined by the combination of d1 and H,h1 and d1 are shown in Fig. 1h1 or d1) are the square lattice. However, considering the change in lattice vectors before and after the operation, h1 corresponds to a 90° counter-clockwise rotation, while d1 corresponds to a shear deformation with a [10] Burgers vector on the (01) shear plane. Note that h1 and d1 are independent, since h1 is a rigid-body rotation while d1 is a shear deformation of the square lattice.In addition, we consider an LID operation described by the following matrix,D. It is clear that such a group is generated by h1 (the generator of the proper rotation group H) and d1 (the LID operation). In fact, it can be proved that D is exactly the SL2(Z) group in this example. Here, SL2(Z) is called the special linear group in 2D, which is represented by 2 × 2 matrices with all entries being integers (Z) and the determinant being 1 . The group SL2(Z), lying discretely on SL2(R), has a role similar to that of Z on R . SL2(Z) is generated by h1 and d1 through matrix row/column operations, the proof of which can be found in textbooks and papers on group theory  do not change the orientation of the crystal lattice. Therefore, they can be applied to the square lattice repeatedly, which leads to the construction of a crystal deformation group D (generated by h1 and d1) can be represented by its Cayley graph G. A Cayley graph  is a colored directed graph that captures the abstract structure of a group . The connections by the red edges capture the global connectivity among the green squares, which conveys the non-local information of graph G (as well as group D).The structure of the group h1 and d1 can be easily figured out, as dictated by the coupling between the crystal symmetry and the LID. For example,From Fig. 2h12d1 = d1h12;(i) h1d1)3 = (d1h1)3 = e;(ii) (h1d1−1)3 = (d1−1h1)3 = h12.(iii) (e.g. the green square) is neglected, we can focus on the deformation of the crystal. Here we consider a partition of the crystal deformation group D. It is clear that the rotation group H is a subgroup of D. Consider an element di of D, and the right coset of H in D is Hdi. All these cosets partition the entire group D into equal-sized non-overlapping sets.H is not a normal subgroup of D, and the right cosets {Hdi:di ∈ D} (i.e.D/H) do not form a group.From a geometric point of view, all four vertices within the same green square in Fig. 2D/H, we consider a graph homomorphism G → HG  are in the same color [Fig. 3a)]. In addition, the vertices in the same color (circled by gray dashed lines) in G become a new vertex in HG and all the green edges in G are removed, while the red edges in G become undirected and unparalleled. In other words, between any two given vertices in HG, there is at most one undirected edge connecting them. The deformation pathway graph HG is shown in Fig. 3b). From a group theory point of view, G includes all the information of group D, while HG captures the structure of D/H . For the convenience of further analysis, we number the vertices in H. GHG is an infinite interconnected graph. Note that each edge in HG could correspond to one or several operations of the form h1md1h1n 3 = (d1h1)3 = e, which is an intrinsic property of the deformation in a square lattice.To visualize the internal structure of HG, we can determine the types of crystalline defect that may possibly be generated during deformation by introducing the geometric compatibility condition. In Fig. 3b), each vertex corresponds to a unique structural state, and the relation between two structural states determines the possible types of defect. For convenience, we define any two vertices directly connected by an edge as the first-nearest-neighbor (1st-NN) vertices and any two vertices connected through another vertex  as the 2nd-NN vertices, and so on. In Fig. 3b), for example, vertices 1 and 2 are 1st-NNs and vertices 2 and 4 are 2nd-NNs. Theoretically, the number of NNs indicates the number of repeated activations of the deformation mode d1. Considering two domains  with a planar boundary, we can determine the types of defect (e.g. dislocation or twin boundary). For the boundary between two domains in the structural states represented by vertices 1 and 2 (1st-NNs) in Fig. 3b), the defects can be determined through the kinematic compatibility condition ], respectively. In this case, F1 = e (identity) and F2 = d1, which are the inputs. b and n are the shear vector and shear-plane normal to be determined, respectively. Q is a rigid-body rotation to be determined in the solution, and F1 and F2 are not unique. For example, F1 can also be assigned as h1, h2 or h3. Similarly, F2 can also be assigned as h1d1, h2d1 or h3d1. It can easily be proved that the choices of F1 and F2 do not affect the solutions of Q, b and n, in terms of crystallographic equivalency.With the deformation pathway graph Q2 is a 53.13° rotation (a 36.87° misorientation in the square lattice), the second solution suggests a Σ5 twin boundary on the (21) plane. The two solutions are illustrated in Fig. 4i.e.F1 and F2, which can be identified in the displacement fields (as well as in the displacement gradient and deformation gradient). In particular, when one domain is within one atomic layer , it becomes a [10](01) type dislocation loop, and the symmetry-breaking process is associated with the motion of the dislocation on Fig. 4.mth-NNs. However, those defects with a large m are usually difficult to generate by a real deformation. The choice of defects in a real crystal system could depend on the loading conditions, e.g. tensile/compressive/hydrostatic stress, strain rate, temperature etc., which dictate the competition between twinning and dislocation. However, we limit our analysis to pure crystallography in this work so that our approach generally applies to all kinds of crystal deformation, without any prior thermodynamic or kinetic knowledge of twinning and dislocations.Similarly, considering the defects between the 2nd-NN vertices, we can apply equation (6)et al., 20042(Z) . However, CDGs are not necessarily special linear groups in general, which will be shown through the following example in 3D. In fact, the CDG of an f.c.c. crystal with a given deformation mode of 1/6〈112〉{111} has to be generated using the generating set presented below, which is not a special linear group (3D lattice group).Note that the CDG is a group description beyond the reach of either point group or space group, as conventionally defined. The CDG takes the information from the LID into account. Geometrically, there are infinite types of LID for a given crystal, because of the translational symmetry. However, there are usually limited types of deformation mode that are energetically accessible from a physical point of view. For example, typical deformation modes in f.c.c. crystals are 1/2〈110〉{111} and 1/6〈112〉{111}, and typical deformation modes in body-centered cubic (b.c.c.) crystals are 1/2〈111〉{110} and 1/6〈111〉{112}. The construction of the CDG depends on the choice of deformation modes, which is a piece of information on the symmetry-breaking pathway during the deformation process. In the example of the square lattice in 2D, a special linear group can be obtained with the deformation mode of 〈10〉{01}. In the literature, special linear groups are also encountered in the definition of the lattice group  in f.c.c. crystals, and a full dislocation can also be captured since a full dislocation is the combination of two Shockley partials. As a result, the typical deformation modes observed in f.c.c. crystals are well described by the following generating set of the CDG:Sf.c.c., the first four elements generate the proper rotational symmetry operations in an f.c.c. crystal, while the last one provides information on the LIDs. Note that the last element in the generating set does not belong to the special linear group SL3(Z), which requires all the entries in the matrix to be integers. As a result, it is clear that the CDG for an f.c.c. crystal is distinctly different from the lattice group of an f.c.c. crystal.Here we consider a deformation mode in an f.c.c. crystal described by the following group generator:e.g. vertex 1), it has six 1st-NNs, 24 2nd-NNs and 84 3rd-NNs. Note that we can find 3-edge circuits and 6-edge circuits in Fig. 5e.g. vertices 1, 2 and 4), there exists a unique 6-edge circuit (through Y) including all three vertices.The DPG for an f.c.c. crystal is shown in Fig. 5b). By utilizing the kinematic compatibility condition . The Σ3 twin between FF3 and FF2 is shown in Fig. 6b). The Σ1 and Σ11 twins between FF4 and FF2 are shown in Figs. 6c) and 6d), respectively. The mathematical details for the determination of the above twins are presented in Appendix Aet al., 2015et al., 1992et al., 1999et al., 2000et al., 2010Some of the above twin structures are illustrated in Fig. 64.Based on the two examples shown above, we present here the general procedure to determine the CDG, DPG and defect structures for a given crystal with a given deformation mode .e.g.h1).(i) Ascertain the point group of the crystal. Determine a subgroup of the point group with all proper rotations (matrix representation) and determine the generators of this subgroup (e.g.d1).(ii) For a given deformation, determine a lattice-invariant deformation matrix with a determinant of 1 (e.g.D).(iii) Using both the generators of the proper rotation group and the lattice-invariant deformation as a new generating set, we can obtain the CDG (e.g.G) with the generating set determined in (iii).(iv) Draw the Cayley graph of the CDG (e.g.HG) through a graph homomorphism.(v) Simplify the Cayley graph to the DPG (mth-NNs using geometric compatibility conditions.(vi) Determine the defect structures between et al., 2006et al., 2006et al., 2011Bet al., 1953The above method for constructing CDGs and DPGs can be applied to any given crystal system. In step (i), the generator of the point group of a given crystal can be found in the Bilbao Crystallographic Server ,


However, intermodular interactions could play a critical role and regulate the activity of the domains and modules in unpredictable ways. Here we investigate in vitro substrate activation by three A domains of the tyrocidine synthetase TycC enzyme, systematically comparing their activity when alone (with the respective PCP domain), in pairs (di-modular constructs) or all together (tri-modular construct). Furthermore, we study the impact of mutations in the A or PCP domains in these various constructs. Our results suggest that substrate adenylation and effects of mutations largely depend on the context in which the domains/modules are. Therefore, generalizing properties observed for domains or modules in isolation should be done with caution.Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that produce bioactive peptides of tremendous structural and chemical diversity, due to the incorporation, alongside the canonical 20 amino acids, of non-proteinogenic amino acids, fatty acids, sugars and heterocyclic rings. For linear NRPSs, the size and composition of the peptide product is dictated by the number, order and specificity of the individual modules, each made of several domains. Given the size and complexity of NRPSs, most  A basic NRPS module is typically composed of an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, and a condensation (C) domain 2. Some A domains require additional proteins called MbtH3 or MbtH-like proteins (MLP)4 for proper function and stability. The activated building block is then attacked by the thiol group of the phosphopantetheinyl (PPE) arm  are modular mega-enzymes that produce peptides independently of the ribosome. For linear NRPSs, the size and composition of the non-ribosomal peptides (NRPs) they produce is entirely dictated by the number, specificity and order of modules they are made ofain Fig. . The A dain Fig. , 1.. Upoarm Fig. , 2. post. 5 Fig. , 0.. Aft. 7 Fig. , 3.. In . 8 Fig. , 4..Figu9, like demonstrated in few successful approaches12.The attractiveness of NRPs for biomedical and biotechnological applications lies in their exceptional chemical and structural diversity, which is associated with the likelihood of finding peptides active in a biological process of interest . This diversity is achieved in numerous ways, some of which include: (i) A domain specificity is not limited to proteinogenic amino acids; (ii) initial C domains can incorporate fatty acids; (iii) auxiliary domains can modify  the building blocks along the way and (iv) the TE domain can release the product as a cyclic peptide. Understanding how NRPSs work is important if we wish to exploit them for synthesizing custom NRPs with novel bioactivitiessub domain is reconfigured by ~48° to complex with ATP and the substrate to perform the adenylation reaction; and, finally, (iii) a thiolation state, in which the C-terminal Asub domain is rotated by ~140° around the large N-terminal Acore to tether the activated substrate to the PPE arm of the PCP domain16. This A domain alternation mechanism is expected to contribute most to the NRP elongation process. At the same time, the PCP domain likely also undergoes some movements as it needs to span the distance between the upstream C domain, the A domain and the downstream C or TE domain, which cannot be solely attributed to the length of the PPE arm (~20 Å) or to the Asub domain movement17. The C domain has also been proposed to exist in an open and a closed state depending on substrate binding18, but the influence of these two states on the overall catalytic cycle of NRPSs remains elusive.During the synthesis process, NRPSs have to undergo large conformational changes to consent to the growing peptide chain to travel along the modules. From studies carried out on individual modules extracted from multi-modular NRPSs, the A domain was reported to adopt three different conformations: (i) an open conformation, where no ligand is bound; (ii) an adenylation conformation, where the small C-terminal A17 or mechanism-based inhibitors22. Thus, each structure represents a staged snapshot of the synthesis process, but the overall movie has yet to be shot. NMR studies are capable of analyzing protein conformational changes in solution, but at least the traditional methods are limited to relatively small proteins and therefore only individual PCP and TE domains or PCP-TE di-domains have been analyzed this way25. Recently, negative stain electron microscopy (EM) images of an A-PCP-C tri-domain construct in complex with a MbtH-like protein (MLP) were compared to a crystal structure of the same protein complex22. The EM images supported the notion that domains within a NRPS module adopt many conformations and do not seem to exist in a set of well-defined states26, in contrast to what was previously proposed27.Insights into the conformational states of NRPSs have been obtained from crystal structures of individual domains as well as multi-domain constructs trapped in a specific state by point mutationsDue to technical limitations and the challenge to express and purify such large proteins, mechanistic studies on multi-modular constructs are lacking. Thus, it is to date unclear how individual modules are structurally and functionally embedded within the context of full-length NRPSs or truncated variants thereof consisting of more than one module. It is licit to ask whether there is an intrinsic communication between modules that ensures proper directionality and progression of NRP synthesis and peptide identity.We specifically asked ourselves whether the adenylation activity of A domains would depend on the context they are embedded in and whether mutations directed towards them or their associated PCP domains would have the same effect regardless of the presence or state of neighboring modules.E. coli without any further codon optimization and without co-expression of a MbtH-like protein. Alongside individual A-PCP di-domain constructs consisting of the last three A-PCP di-domains of TycC , we built two di-modular constructs (TycC8-9 and TycC9-10) and one tri-modular construct (TycC8-9-10). All these constructs were either kept “wild type” or mutated in each A and PCP domain (one at a time). To systematically compare A domain activity we employed a PPi release assay28 which has been recently adapted to the study of NRPSs31. The advantage of this assay is that the detection is spectroscopic and does not require radioactivity. Our findings suggest that the context into which the A domains are embedded does play a critical role in regulating their activity, likely because modules in NRPSs interact in complex ways that are not easy to predict by knowing the structure and function of the individual components.To this aim, we constructed a library of 34 constructs based on the very well-studied tyrocidine synthetase TycC Fig. , which c32 to locate the A and PCP domains of the last three modules in TycC  and (2)), TycC9 ((1) and (2)) and TycC10 ((1) and (2)). As BAP1 cells express the chromosomally integrated promiscuous PPtase Sfp35, we expect the PCP domains in the purified proteins to be loaded with the PPE arm, as previously reported37. Interestingly, we found that with the new start site identified in this study the adenylation activities of the A domains were more comparable, while the previously established start site led to higher variability 38 to alanine , since in this case lysine was accepted more robustly ), we then constructed two di-modular constructs: one consisting of modules 8 and 9 (we refer to this construct as TycC8-9) and one containing modules 9 and 10 (which we refer to as TycC9-10) Fig. . Note thlls Fig. , then anTycC8-9, with and without TE domain, showed activation of valine and ornithine, regardless whether the amino acids were introduced separately or simultaneously Fig. . The prePCP*) and the PCP mutant of module 10 in the di-modular TycC9-10 + TE construct (TycC9-10PCP*) served as negative controls. In agreement with the kcat values for TycC9-10 + TE obtained in the online PPi release assay, the radioactive signal when ornithine and leucine were present was as high as that for the positive control . The mutants were expressed in and purified from lds Fig. . Mutatiolds Fig. . These rlds Fig. , albeit lds Fig. . This islds Fig. . Mutatiolds Fig. , as prevlds Fig. . To testine Fig. . We founine Fig. . Surprisine Fig.  and S5a.E. coli cells with lower yields compared to the other constructs  had, moreover, a similar negative impact also on TycC9-10ΔTE . These mutants, as well as the wild type protein, were purified from cts Fig. . Mutatincts Fig. , in goodcts Fig. . The PCPΔTE Fig. . UnfortuΔTE Fig. . Howeverine Fig. .E. coli cells with good yields despite its very large size of about 326 kDa .NRPSs are fascinating enzymes that attract the attention of scientists from different disciplines due to the enormous benefit that would derive from the ability to re-engineer them to produce desired peptides. While exciting advances have been recently made towards finding rules to cut and paste modules in a defined manner to build functional chimeric NRPSsThere are three sets of data pointing to this conclusion derived from measuring A domain activity for different constructs with different substrate combinations in otherwise identical conditions: (i) effects of mutations; (ii) specificity of A domains; and (iii) activity of A domains.A*-10 + TE), it is likely that the mutation nonetheless affects other aspects of A domain functionality, such as successful communication with the PCP domain. Indeed, while we cannot exclude that the tripeptide is made at levels below those we can detect under these conditions, the aspartic acid we mutated is conserved in all A domains45, regardless if starter or elongation ones, indicating that a compensation of the mutation supporting peptide production is unlikely. Another explanation is that the adenylation activity we measure is that of another A domain present in the multi-modular construct. As a matter of fact, the values obtained in the online PPi assay cannot be assigned to any specific A domain. This would suggest that the point mutation changes the specificity of neighboring A domains, for instance due to a different conformation assumed in the absence of ATP but presence of the substrate16. The current experimental techniques at our disposal do not allow us to clarify this issue.Mutating the ATP binding site of the A domain has the expected effect of abolishing adenylation only when applied to the excised A-PCP di-domains Fig. . Howeveri, which we measure – independent of the next step, which is its attachment onto the PPE arm. Indeed, other studies have used excised A domains where the PCP domain was absent all together and they could measure A domain activity, albeit it proved to be lower than in the presence of the PCP domain46. However, the same PCP domain mutation impairs the activity of the cognate A domain in the di-modular constructs . This is exemplified by the inability of TycC9-10 to repeatedly activate leucine in the absence of ornithine Fig. . This isIn conclusion, we believe that generalizations for domain and modules of NRPSs should be done with great caution, as their behavior appears to be very much dependent on the context they are embedded into.48 using the Gibson Assembly® Master Mix  according to the manufacturer’s instructions. The pTrc99a vector49  served as a backbone for the insertion and expression of the different constructs. In short, the backbone and TycC inserts were amplified using the Phusion Flash High-Fidelity PCR Master Mix  from the full-length vector and a glycerol stock of Brevibacillus Brevis ATCC 8185 , respectively. In TycC9, specific for Orn, we detected several SNPs in comparison to the genomic sequence deposited in the NCBI database43. A domain: G190(ggg) → E(gag) and T201(acc) → I(atc). PCP domain: L35(ctt) → I(att). C domain: V6(gtc) → V(gtt) silent, V43(gta) → V(gtg) silent and A229(gcg) → E(gag). All primers (Sigma Aldrich) were designed for the DNA fragments to have an overlap between 15 and 25 base pairs which serve as a complementary recognition and fusion site during Gibson Assembly  with the promiscuous PPtase Sfp integrated into their genome34 . A large expression culture (250–400 mL of LB-Miller containing 0.1 mg/mL ampicillin) was inoculated by adding 1:500 dilution of overnight culture and grown at 37 °C at 180 rpm until it reached an OD600 of 0.7–0.9. IPTG  was then added to the culture to a final concentration of 0.5 mM, and the culture was further grown at 18 °C overnight. The next morning, cells were harvested via centrifugation at 5 000 × g for 30 minutes. The cell pellet was resuspended in lysis buffer  and stored at −80 °C for 12 or more hours. Cells were lysed via sonication  and cleared at 15 000 × g for 30 min. The supernatant containing the soluble protein fraction was incubated with 1 mL Ni-NTA beads  for 2 hours at 4 °C. The loaded Ni-NTA beads were washed twice with three column volumes of lysis buffer via gravity flow before being eluted with elution buffer . Eluate fractions were checked for protein content via Ponceau S staining. The fraction containing the major part of the protein was concentrated and buffer exchanged to storage buffer  in spin columns  with the appropriate MWCO. Proteins were stored at −20 °C until use.Plasmids harboring the desired TycC constructs were transformed into BAP1 cells for protein expression and i release assay was performed as described before by Kittilä et al.30. In short, all components  is the decrease in absorption at 340 nm calculated fitting the curve in the linear range; ε340(NADH) is the molar extinction coefficient of NADH (ε340(NADH) = 6 220 M−1 cm−1)52; d is the path length (~0.2 cm in a well of a 96 well plate filled with 100 µL reaction volume); and cenzyme is the enzyme concentration (in µM).The online PPts Table  were comcat value of three measurements was calculated and background subtracted by the mean value of the negative control. The error was determined by subtracting the standard deviation (SD) of the negative control from the SD of sample, including error propagation:For each enzyme-substrate combination, the mean k7. Enzymes were mixed at 0.5 µM in a total volume of 100 µL TRIS-HCl (pH 7.5) together with 10 mM MgCl2, 2 mM TCEP, 2 mM L-Orn, 10 µM L-Leu ). The reaction was started by adding 5 mM ATP and quenched after 1 min with ice-cold 800 µL TCA (10% trichloroacetic acid solution). Proteins were coprecipitated with 15 µL ice-cold BSA (25 mg−1ml−1 solution) and pelleted for 30 min at 13 000 rpm. The pellets were washed twice with 800 µL ice-cold TCA and resuspended in FA (10% formic acid solution). After addition to 3 mL scintillator liquid, the amount of radiolabeled leucine covalently attached to the PPE arm of the respective enzyme was measured in a scintillation counter (Beckman Coulter LS 6500 Liquid Scintillation Counter).Thioester formation of radiolabelled leucine was investigated according to a modified protocol2, 2 mM TCEP, 100 µM L-Val, 100 µM L-Orn and 100 µM L-Leu and incubated at 37 °C. With the addition of 5 mM ATP the reaction was started and after 1 h quenched with 10 µL of formic acid. The samples were pelleted , the supernatant mixed with 20 µL acetonitrile and 10 µL were subjected to the LC-ESI-MS (Agilent 6130B Single Quadrupole). The MS was used in positive mode together with a NUCLEODUR® HILIC column . Solvent A contained H2O, 0.15% FA and 10 mM ammonium formate and B H20, 95% acetonitrile, 0.15% FA and 10 mM ammonium formate. Extracted ion traces of the peptide masses were recorded.The formation of the respective peptide products was analyzed by LC/MS. Enzymes (3 µM) were mixed in TRIS-HCl buffer  with 10 mM MgClSupplementary Information


As the highest elevation permafrost region in the world, the Qinghai-Tibet Plateau (QTP) permafrost is quickly degrading due to global warming, climate change and human activities. The Qinghai-Tibet Engineering Corridor (QTEC), located in the QTP tundra, is of growing interest due to the increased infrastructure development in the remote QTP area. The ground, including the embankment of permafrost engineering, is prone to instability, primarily due to the seasonal freezing and thawing cycles and increase in human activities. In this study, we used ERS-1 (1997–1999), ENVISAT (2004–2010) and Sentinel-1A (2015–2018) images to assess the ground deformation along QTEC using time-series InSAR. We present a piecewise deformation model including periodic deformation related to seasonal components and interannual linear subsidence trends was presented. Analysis of the ERS-1 result show ground deformation along QTEC ranged from −5 to +5 mm/year during the 1997–1999 observation period. For the ENVISAT and Sentinel-1A results, the estimated deformation rate ranged from −20 to +10 mm/year. Throughout the whole observation period, most of the QTEC appeared to be stable. Significant ground deformation was detected in three sections of the corridor in the Sentinel-1A results. An analysis of the distribution of the thaw slumping region in the Tuotuohe area reveals that ground deformation was associated with the development of thaw slumps in one of the three sections. This research indicates that the InSAR technique could be crucial for monitoring the ground deformation along QTEC. Permafrost, defined as soil or rock ground that remains frozen (ground temperature below 0 °C) for two or more consecutive years ,2, has tTraditional geodetic measurement methods such as levelling and the global position system (GPS) surveys, can achieve high-precision monitoring. However, these point-based geodetic measured methods are limited to discrete points on fixed routes and are time consuming. Compared with those methods, the satellite remote sensing provides a valuable tool for observing large and hard-to-access areas with high spatial and temporal resolution . SynthetTo mitigate the intrinsic limitations of the traditional differential InSAR (DInSAR)  , time-seDue to the merits of time-series InSAR, many studies have used it to retrieve surface deformation information related to permafrost thawing and freezing in QTP ,37,38,392 area [The studies listed in 2 area . Rouyet 2 area . CombiniThe objectives of this paper were to retrieve the surface deformation along QTEC from the Wudaoliang to the Tuotuohe section over a 20-years period using time-series InSAR technique and to analyze temporal evolution of the QTEC deformation. More than 90 SAR images, including ERS-1, ENVISAT, and Sentinel-1A, were collected to jointly retrieve the feature of ground deformation from 1997 to 2018. A hybrid time-series methodology taking advantage of the merits of PSI and SBAS was used to identify more measurement points . MoreoveThe permafrost region along the Wudaoliang-Tuotuohe section of QTEC was chosen as the study area. The area is in the in the Hoh Xil mountain area between the Kunlun Mountain and Tanggula Mountain ranges and is the source area of the Yangtze river, which is in the northern part of the QTP . The QTRWithin the QTEC coverage, several key developmental projects have been constructed, such as QTR, QTH, and electric transmission power line d–f. Due To reveal the ground deformation in the study area in the selected 20-year period, SAR images from three different satellites were collected. There are ERS-1 SAR images acquired from October 1997 to December 1999; ENVISAT SAR images from November 2004 to July 2010, and Sentinel-1A SAR images from April 2015 to December 2018. The coverages of the above SAR stacks are shown in Studies have demonstrated that the main challenges and limitations of the InSAR technique in detecting the ground deformation in the permafrost region are the serious temporal decorrelation and non-linear deformation trends caused by the seasonal thaw-freeze process of active layer ,37,46,47Firstly, all the SAR images were co-registered. Then, a multi-temporal InSAR data processing strategy was used to retrieve ground deformation. Considering the different attribute of SAR stacks with different wave lengths, different small baseline strategies were adopted for those SAR stacks . ThroughThe thaw-freeze process of the active layer is complex and correlated with many factors, such as vegetation, snow, soil moisture, soil properties and temperature . In permR is the long-term deformation rate, tA and fA are the thawing and freezing deformation coefficients, respectively; and ADDT and ADDF are the accumulated degree days of thawing and freezing, respectively. ADDT reaches its the maximum at the end of the thawing season. The daily ADDT and ADDF were calculated based on the air temperature measured at the Wudaoliang Meteorological station. Due to the sporadic acquisitions of ERS-1 images, a deformation model without a seasonal term was used for ERS-1 datasets.The deformation model is defined as follows:T (t) is the temperature on day t. The thawing and freezing onsets of the active layer are fixed as 1 May and 15 September, respectively ,37. Howe2) > 0.9 and root mean square error (RMSE) < 2.7. CPs are those points with high coherence and stable amplitude value during the whole observation period. In permafrost area, the ground feature includes four types: permafrost engineering, Thermokarst Lake, alpine meadow, alpine desert . In ordeε is the random phase error, ai represents the estimated parameters. The interferograms with obvious phase ramps were corrected using this model. After that correction process, we assumed that most of the topography related phase errors (DEM error and atmospheric delay) have been removed.The atmospheric delay is influential in high latitude mountainous regions. Our study area has an average elevation of over 4400 m with some mountains. In the mountainous areas, the stratified troposphere can produce serious atmospheric delays in the interferograms. Obvious residual orbital phase was in some interferograms. In this paper, to remove those phase ramps, we applied a phase ramps correction model combining a biquadratic model for orbital phase ramps and a linear model for elevation dependents errors :(3)φ method . The atmAfter identification of the CPs, all the CPs were connected to further remove the effects of the atmospheric delay. The differences of those differential interferometric phase between the neighboring CPs in the ith interferograms can be written as:R, A, and R and A are along the slant light of sight (LOS) direction. We assumed that the detected deformation is in vertical direction, and the LOS estimated deformation was converted to the vertical direction by dividing the cosine of the average incidence angles. The specific procedures of the approach are illustrated in The identified CPs were firstly connected based on the Delaunay triangulation network. Then, the differential phase of all the edges were calculated, which is beneficial to further remove atmospheric and orbital errors. The parameters approach . After tUsing the time-series InSAR method described above, the estimated average ground deformation rate along QTEC from the Wudaoliang to Tuotuohe sections using three C-band SAR stacks from 1997 to 2018 have been generated, including the deformation rate from 1997 to 1999 calculated with ERS-1 data see a, the deThe ground deformation rate along the QTEC ranges from −10 to +10 mm/year during the 1997–1999 observation period derived from ERS-1 data. For the ENVISAT and Sentinel-1A experiments, the estimated deformation velocity was primarily in the range of −20 to +10 mm/year. The spatial distribution of the deformation before 2004 was quite different from those after 2004, and the deformation rate of the ERS-1 was inaccurate due to the few SAR datasets and heterogeneous spatial-temporal baseline.In this study, we choose the QTR as an example to analyze the deformation of permafrost engineering. Obvious deformation along QTEC was detected in three areas as enclosed by the red dashed ellipses in The Beiluhe basin region is in the tundra of Hoh Xil and is underlain by cold permafrost. The terrain is relatively flat and most of the slope is less than 40°. The soil moisture content in the surface is high in the summer season and can reach 0.3 . The vegDuring the field investigations in 2014 and 2015, some surface cracks or fissures of about 20 cm along the QTEC and alpine meadow regions were found in the Beiluhe regions, as shown in The Fenghuo Mountain, with an average elevation of more than 5000 m, is to the southeast of Hoh Xil, 380 km away from the city of Golmud. The 1.33 km long QTR Fenghuo Mountain tunnel was successfully traversed on 19 October, 2002. The average elevation of Tuotuohe region is about 4780 m. The ALT ranges from 1 to 4 m. In the last 2010s, the permafrost in QTP underwent serious degradation due to global warming. During the period from 1961 to 2007, the observed air temperatures over the QTP showed a rising trend, with a mean increasing rate of 0.037 °C/year . Against°C/year . Lu et a°C/year  proposed°C/year  is the sThe modeled MAGT of the study site is shown in Thermokarst lakes have been developing along the QTEC as a result of increased human activity and ongoing climate warming . The the2, respectively . In the mountainous areas, the deformation rate is small and stable. More SAR images with short revisit cycle are needed in the future research.(1)Before the operation of the QTR, the QTEC from Wudaoliang to Tuotuohe was in stable with a deformation velocity of less than −5 mm/year from ERS-1 images. The embankment of the engineering structure was considered stable. The thawing and freezing of the active layer were the main deformation driving-forces. After the QTR started operation and the human activities increased, some sections of the QTEC were underwent obvious deformation, and the deformation has increased more recently.(2)From 2015 to 2018, obvious deformation was found in three areas: Beiluhe, southern of Fenghuo Mountain, and Tuotuohe, with the large deformation rates of over −20 mm/year. Real-time deformation monitoring must be conducted in these sections. The subsiding areas are consistent with the permafrost areas with large MAGTs.(3)This work demonstrated the potential of the time-series InSAR for the surveillance of the state of QTEC on a large scale. Interferometric decorrelation is still one of the problems for time-series InSAR monitoring of the ground deformation in permafrost region. With the proposed innovative methods and newly-launched SAR systems with shorter revisit cycles (Sentinel-1A/1B and TerraSAR-L), higher temporal sampling allows us to better characterize the ground deformation related to the process of permafrost thawing and freezing.In this paper, we presented an application using the time-series InSAR technique with multisensory SAR datasets to monitor the permafrost ground deformation along the QTEC from 1997 to 2018. A deformation model combining a linear subsidence term and seasonal deformation term was adopted in the time-series InSAR method to exploit the permafrost ground deformation. Three deformation rate maps along a 100 km stretch of the QTEC were generated from 9 ERS-1, 39 ENVISAT, and 41 Sentinel-1A images. The three independent InSAR measurement results showed a consistent deformation trend and most of the ground surface along the QTEC was stable with the deformation rate ranging from −10 to 10 mm/year. The conclusions are summarized as follows:In future work we will focus on investigating the three-dimension deformation in permafrost regions using multiple satellites SAR images, and retrieving the geophysical parameters of permafrost such as the active layer thickness, on a larger scale.


Phytophthora infestans, the causal agent of potato late blight, is without a doubt the most (in)famous. Other important plant pathogenic oomycetes include downy mildews and Albugo [Saprolegnia, Aphanomyces, and Pythium) primarily affect marine organisms such as fish and crustaceans but can also cause harmful diseases in humans and cattle. Here, we outline unique features of oomycetes and their cellular signaling systems. We focus on one particularly interesting class of signaling components, the so-called GPCR-bigrams, in which a typical signaling domain is preceded by a G-protein coupled receptor (GPCR) domain and propose future directions to elucidate the function and activity of this exceptional group of signaling proteins.Oomycetes are diverse eukaryotic microorganisms, comprising important pathogens as well as free-living saprophytes . Plant pd Albugo . Animal Oomycetes are easily mistaken for fungi but are different in many ways. At the organismal level, morphology, growth pattern, and mode of dispersal are shared, and the 2 groups occupy similar ecological niches. Only at the cellular and molecular level do the differences become apparent. For example, the cell walls have a different composition; whereas chitin is the main component in fungi, oomycete cell walls consist of cellulose and β-glucans . OpposedObviously, these differences also have consequences for the efficacy of chemical control agents. For example, a major class of fungicides broadly used in healthcare and agriculture are sterol biosynthesis inhibitors (SBIs), but since oomycetes do not synthesize sterols, they are insensitive to SBIs. The quest for novel compounds for controlling pathogenic oomycetes is a continuous challenge. We postulate that oomycete-specific cellular signaling components, such as GPCR-bigrams, hold potential as novel drug targets. Likewise, due to their central role in development and pathogenicity, fungal GPCRs have recently been proposed to be druggable targets for novel antifungal agents .Phytophthora has several protein kinases and phospholipid kinases with accessory domains that are normally not found in combination with kinase domains. On the other hand, 2 ubiquitous enzymes, i.e., protein kinase C (PKC) and phospholipase C, seem to be absent, at least in their stereotypical forms [Cellular signaling in oomycetes is inscrutable and holds many novelties. Several oomycete signaling components are clearly distinct from their homologs in organisms from other taxa. Many of these are composed of unique combinations of protein domains not known to exist in other organisms , 9. For al forms , 9.One particularly interesting class of unique signaling components comprises the GPCR-bigrams. These proteins have an N-terminal GPCR domain typically composed of 7 transmembrane (TM) regions, combined with a C-terminal catalytic accessory domain . Based oThe predicted catalytic activity of these GPCR-bigrams is unique. There are other examples of GPCRs with accessory domains, such as adhesion GPCRs, but their extracellular N-terminal extensions have a role in protein–protein interaction and are not predicted to have catalytic activity . Plants P. infestans has 4 GPCR-ACs in addition to 8 canonical adenylate cyclases (ACs) (http://fungidb.org/fungidb/app/record/organism/NCBITAXON_403677). This underscores the importance of GPCR-bigrams for oomycetes. In case the catalytic domain in a GPCR-bigram would not have an advantage over the canonical enzyme, there would be no evolutionary pressure for the GPCR-bigram to sustain. Thus, the strong conservation of GPCR-bigrams throughout oomycetes indicates that having GPCR-bigrams is advantageous. Clearly, oomycetes need GPCR-bigrams, but for what purpose?In oomycetes, GPCR-bigrams occur next to regular conserved enzymes. For example, RpkA in the slime mold Dictyostelium discoideum displayed defects in cell density sensing, bacterial defense, and phagocytosis and had reduced phospholipid levels [P. infestans resulted in aberrant asexual development and reduced pathogenicity [Phytophthora sojae transformants with a silenced GPCR-PIPK gene showed similar phenotypes but, in addition, showed reduced chemotaxis toward soybean root tips and the soybean isoflavone daidzein [The conservation of all GPCR-bigram types in oomycetes and the presence of GPCR-PIPKs in several unicellular eukaryotes almost indisputably suggests that they are functional proteins. To date, however, there are only a few studies addressing their biological role, and these are essentially limited to GPCR-PIPKs. Knockout lines of the single GPCR-PIPK gene d levels , 14. Silgenicity . Phytophdaidzein . These lThe catalytic domains of GPCR-bigrams are usually the core domains in effector proteins regulated by G-protein signaling. Hence, it is conceivable that GPCR-bigrams provide a direct link between GPCR sensing and catalytic activity. Below, we speculate how GPCR-bigrams may transduce signals.An intriguing possibility is that the catalytic domain is activated directly upon binding of an agonist  to the GPCR domain  or afterAnother possibility is that the GPCR domain activates heterotrimeric G-proteins that then stimulate the activity of the catalytic domain . LikewisGPCRs can display biased agonism, a phenomenon signifying that different ligands can induce specific receptor profiles on the same receptor molecule . Such prP. infestans has only a single GRK gene and lacks PKC [P. infestans phosphorylates GPCR-bigrams to initiate β-arrestin recruitment.Besides heterotrimeric G-proteins, β-arrestins can also act as molecular switches transmitting GPCR-sensed signals. Initially, β-arrestins were thought to serve a main role in the desensitization of GPCRs, initiating the internalization of an activated GPCR. Later, it was recognized that β-arrestins can facilitate signal transduction to mitogen activated protein kinases (MAPKs) by serving as scaffolds to recruit proteins to an activated GPCR . The phoacks PKC . It is cMost if not all GPCRs are activated upon recognition of an external signal. Likely, the receptor domains of GPCR-bigrams are also capable of recognizing a ligand, and obvious questions that arise are the following: what is the nature of these ligands and how can they be identified? So far, the only putative candidate is the isoflavone daidzein . There i2+ using biosensors or induction of reporter expression [As GPCRs are important drug targets in human medicine, ligand discovery is primarily focused on human GPCRs. A common and popular approach is screening (human) cells expressing the GPCR of interest with chemical libraries and monitoring changes in production of second messengers such as cAMP, IP3, or Capression . As yet,Phytophthora. Alternatively, the GPCR-bigrams could be expressed in yeast, taking advantage of the many available signaling mutants and reporter strains. Also, yeast is easy to manipulate and contains only 2 endogenous GPCR-signaling systems that can be eliminated [Though the setup would be artificial, one could envision expressing an oomycete GPCR-bigram in a mammalian cell line and then screening a chemical library of known compounds or mixtures comprising putative ligands, such as exudates from plant tissue or from iminated . Of the P. infestans (http://fungidb.org/fungidb/app/record/organism/NCBITAXON_403677), we assume that arrestins have a role in oomycete cellular signaling somehow. For analyzing β-arrestin desensitization and recruitment by GPCRs, several assays are available. Some are based on human arrestin fused to a protein that upon activation induces reporter gene expression  or β-galactosidase activity  [As yet, oomycete β-arrestins have not been studied, but given the presence of 3 proteins with arrestin domains in n assay) , 22. Othn assay) , for whiPhytophthora sojae [With the recent development of a CRISPR/Cas9 system in ra sojae , it mighOur current understanding of how GPCR-bigrams function is very limited. The challenges that lie ahead are determining their role in cellular signaling and their biochemical mode of action and answering the question why oomycetes have such unique signaling proteins. What is the advantage of having GPCR domains linked to catalytic accessory domains? Does it provide, for example, shortcuts for more efficient signaling? This could be the case if the catalytic domain is under direct control of the GPCR domain, a situation that is unprecedented. Another major challenge is to identify ligands recognized by GPCR-bigrams and to determine how such ligands can be exploited. We envision that profound knowledge of these enigmatic signaling components and their ligands exposes new strategies for designing novel, oomycete-specific control agents to mitigate damage caused by these devastating pathogens.