Our results show that a single MMF based deformation sensor is excellent in terms of system simplicity, resolution and sensitivity, and can be a promising candidate in deformation monitoring or shape-sensing applications.Optical fiber bending, deformation or shape sensing are important measurement technologies and have been widely deployed in various applications including healthcare, structural monitoring and robotics. However, existing optical fiber bending sensors require complex sensor structures and interrogation systems. Here, inspired by the recent renewed interest in information-rich multimode optical fibers, we show that the multimode fiber (MMF) output speckles contain the three-dimensional (3D) geometric shape information of the MMF itself. We demonstrate proof-of-concept 3D multi-point deformation sensing via a single multimode fiber by using  Optical fiber sensors provide an efficient and competitive sensing solution, owning to their unique properties, including light-weight, low cost, freedom from electromagnetic interference, and resistance to harsh environment and chemicals11. Among optical fiber bending sensors, fiber Bragg gratings (FBGs)14, long period fiber gratings (LPFGs)16 and specially designed optical fibers18 are popular choices owing to their robustness. For example, the fiber sensor presented in Ref.19 exhibits high sensitivity (up to 1270 pm/°) and can be used to monitor small bending angles (2°). In Ref.20, a two-dimensional bending sensor based on long period gratings (LPGs) fabricated in three-core fibers were demonstrated. Furthermore, FBGs were fabricated in a six-core fiber to achieve a spatial resolution down to 40 µm21. While these existing fiber-optic sensors have demonstrated good sensitivity and resolution, the application of these previous optical-fiber-shape sensors are limited by the complex fabrication process and expensive detection systems. The fabrication of high-accuracy FBGs and good-performance multicore fibers is complicated. The signal processing and interrogation systems involved are bulky and high cost, which especially limits their application in wearable and portable devices. Furthermore, the spatial resolution of fiber-grating-based bending sensors is restricted by the minimal length of individual gratings that need to be incorporated to measure each deformation point. Therefore, a deformation sensor consisting of only standard and low-cost fibers would be ideal.Complex deformation, bending or shape sensing has received growing attention, due to their booming applications in interactive sensing devices24. The MMF output speckles contain rich information and have been used in applications such as spectrometers25, optical trapping26 and imaging27. Very recently, utilizing MMF output speckles, researchers employed deep learning in input-pattern reconstructions, including MINIST characters29, natural scenes30 and even randomly distributed spatial information31. Here, we hypothesize that the MMF speckles possess the information that relates to the complex deformation along the fiber itself. In this work. We attempt to use machine learning to interpret the complex deformation of the fiber with output speckles. In fact, speckle-based MMF bending classification was recently reported34 on one-point low dimensional to classify such MMF bending. In Ref.34, a spring needle was used to press the fiber to achieve bending at one point, where the freedom of deformation is relatively low as both ends of the fiber were fixed. In this work, we propose the following to experimentally study speckle-based 3D multi-point deformation classification through a single MMF.Multimode fibers (MMFs) are high-capacity information channelsFirstly, we studied the bending-location classification along the MMF axial direction using MMF speckles. As shown in Fig. k-nearest neighbors algorithm (KNN) are shown in Fig. Figure We then set up an electrical-motor driven system to test the classification of different bending angles at one deformation location on a piece of MMF Fig. a. A 20 cThe results presented above suggest: (i) the deformation applied at different points along the same MMF results in different speckle patterns and the machine learning algorithms (i.e. KNN here) is able to distinguish such subtle differences; and (ii) different degrees of deformation at the same point also result in different output speckles, which can subsequently be classified by KNN. These forms a good basis of the following complex deformation sensing experiments.Next, we tested the performance of our sensor for two-dimensional (2D) multi-point bending. We used a 23 cm long MMF and fixed 12 cm section of the fiber as the experimental deformation region  for the proof-of-concept demonstration of three-dimensional (3D) deformation sensing. We first attached a 5 cm section of a 12 cm long MMF to a 5 cm-long end-effector section of the robotic arm for 3D one-point deformation test, as shown in Fig. Based on the 3D one-point deformation results above, we tested 3D three-point deformation sensing using a 220 cm-long MMF by attaching a 43 cm section of the fiber onto the entire robotic arm and covering three bendable joints, as shown in Fig. 29, or to update the neural network continuously by semi-supervised learning31.One foreseeable challenge when applying the single MMF deformation sensor in real world is the high variability of the MMF output speckles, which is sensitive to the environment conditions, including temperature, vibration, and mechanical stress. Any small changes in these can lead to variations of the speckles, as shown in Supplementary Most experiment results presented in our work show high classification accuracy of 100% or very close to 100%. We believe that those observe classification errors  are not the inherent error of the MMF bending sensor itself but are mainly due to the experimental setup, including the errors of the motor, the elasticity of the fiber jacket material after being stretched, and the small but undesirable relative movements between the taped-fixed fiber and the robotic arm in each robotic arm movement. Again, these can be improved with a specially designed packaging to secure the fiber on the structure or device to be monitored.34. Thirdly, 3D multi-point deformation is achieved via a single standard MMF and this is expected to be extended to more complex deformation or 3D shape sensing. In comparison, FBG based 3D shape sensing requires multiple fibers or specially designed multicore fibers. The advantages of our MMF deformation sensor include: high degrees of freedom, simple detection setup without requiring additional wavelength interrogation devices, and an overall low-cost system. This single MMF based 3D multi-points deformation sensing technique may find applications in limb motions monitoring, surgical tools manipulation, wearable device sensing and robotic control.In summary, we have demonstrated 3D multi-points deformation sensing via a single MMF using machine-learning based speckle classification. The demonstrated single MMF deformation sensor is advantageous compared with traditional optical fiber deformation sensors in several aspects. Firstly, the transducer itself is a standard MMF and is therefore extremely low cost. The sensing system can be incorporated as a compact and wearable device where only a light source and an image sensor are needed, without requiring complex wavelength interrogation systems such as those needed for FBG sensors. Secondly, our single MMF deformation sensor can be applied to various types of deformation and a good resolution is achieved. As shown in our experiment, a 0.3 mm spatial resolution is achieved in Fig. 35. A longer MMF increases the variations between speckles for the same deformation but at the same time introduces more instability. The other end of the MMF was imaged with a digital CMOS camera  through a 30 mm focal lens. All fibers were bare fibers and were tested without protective jacket or connectors. How the optical fiber speckle changes with deformation is given in Supplementary Output from a laser pointer  was coupled into MMFs  of different lengths between 110 and 2200 mm according to the requirements of different bending experiments). It has been experimentally demonstrated that multimode fibers with larger cores or smaller numerical apertures are more sensitive to deformation38. KNN is simple and requires tuning only one hyperparameter , while neural network training involves many hyperparameters controlling the size and structure of the network as well as the optimization procedure. For advanced deep neural networks such as convolutional neural network (CNN), they require a much larger size dataset. Due to the small dataset size in our experiment, KNN is ideally positioned to classify the speckles. Another important justification is that compared it with deep neural networks, such as CNN, KNN can obtained a similar accuracy in our experiment dataset. The results are provided in Supplementary The KNN algorithm was used to analyze the experimental data owing to its simple implementation and good classification performancek nearest neighbors determines its class assignment by either averaging the class numbers of the k nearest reference points or by obtaining a majority vote for them.The KNN classification is performed by using a training dataset which contains both the input and the target variables and then by comparing the test data which contains only the input variables to that reference set the distance of the unknown to scaler.fit_transform function within Python sklearn.preprocessing Library. Bending state labels were inserted in each figure and at the same time corresponding speckle patterns of different bending states were identified. Subsequently, the labels and their corresponding speckles were set up in a dataset. This dataset was then split into two data groups for training and testing, respectively. After the training of KNN, speckle images in the test dataset were sent to KNN to obtain the predicted bending-state labels. The predicted labels were compared with the original bending state labels to determine the accuracy of KNN classification.The speckles were firstly normalized and standardized before applying them with the KNN. The size of the speckle was set to 120 × 120. Image preprocessing was done by using k. The best model hyperparameters is k equals to 3. The distance metric for the proximity evaluation in this paper was chosen to be the Euclidean distance.The kNN algorithm involves three main factors: the training set, the distance measurement, and the size of Supplementary Information.


This study describes the structure of the chloroplast ribosome-associated molecular chaperone trigger factor at 2.6 Å resolution. It is shown that this eukaryotic trigger factor has evolved specific structural features in plants that are distinct from those of the bacterial homolog, contributing to a better understanding of co-translational protein folding in plastids. cis–trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Å resolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl  Ribosome binding is mainly mediated via the conserved GFRxGxxP signature motif and two surrounding α-helices of TF contacting ribosomal proteins Rpl23 and Rpl29 as well as domain III of the 23S ribosomal RNA  with sequence homology to FK506-binding proteins (FKBPs). The PPIase constitutes the so-called head, opposite the RBD, in the three-dimensional structure of TF. The PPIase allows TF to actively catalyze the cis–trans conversion of peptidyl-prolyl bonds in vitro  and from Arabidopsis thaliana (Arabidopsis) demonstrated that the trigger-factor proteins have maintained the dragon-shaped conformation; however, their domain orientation seems to vary when compared with the conformation of TF from E. coli , whereas algal TIG1s show greater sequence variance in this region, with only the first glycine and the last proline being conserved in the motif. Interestingly, the algal trigger-factor binding site on the ribosomal Rpl23 protein shows equal sequence variance at this site, which points to co-evolution of the trigger-factor ribosome-binding interface in these phyla  at the atomic level. We resolved the X-ray structure of truncated CrTIG1 with the PPIase domain and the C-terminal SBD. The structure shows an uncommon tilted PPIase domain which interacts intermolecularly with the SBD over a coiled-coil motif. The SBD does not contain the common ‘open arms’ features of the bacterial TF structure, but rather a conformation with aligned arms. The inner part of the cave built by the arms together with the PPIase domain shows a very negatively charged surface. The PPIase domain exhibits a low but measurable PPIase activity.The ribosome-binding signature motif of plastidic TIG1 is highly conserved within the land plants lineage  was produced in E. coli using a modified two-step protocol as described by Guerrero et al. (2001−1), grown for 16 h, harvested and washed with sterile deionized water. The cells were then diluted in 2 l SelenoMethionine medium (Molecular Dimensions) supplemented with the same antibiotic and 40 µg ml−1l-selenomethionine (Molecular Dimensions) to an OD600 of 1.0. After incubation for 30 min at 303 K, CrTIG1-SeMet was expressed by the addition of 0.4 mM isopropyl β-d-1-thiogalactopyranoside for 17 h at 290 K. CrTIG1-SeMet was purified as described for the native construct, except that 1 mM DTT was maintained in all buffers subsequent to elution from the chitin matrix. al. 2001. A precu2.2.CrTIG1 were concentrated to 23 mg ml−1. Initial crystals were obtained in 96-well plates by the sitting-drop vapor-diffusion method at 291 K using the automated crystallization facility at the Department of Structural Biology, Saarland University  and reservoir solution were mixed and equilibrated against the reservoir solution. Crystals appeared between 10 and 90 days and were further optimized in 24-well plates using the hanging-drop vapor-diffusion method. Crystals were obtained at 277, 283 and 291 K in various crystallization conditions. Only crystals that grew at 283 and 291 K could be used for diffraction experiments; they diffracted to between 2.6 and 6 Å resolution. The crystals obtained at 277 K were very fragile and did not tolerate any handling or cryoprotectant. Crystals growing at 283 or 291 K tolerated glycerol over PEG 3350 as the best cryoprotectant. High-resolution structural information was obtained for native and selenomethionine-labeled proteins, which were crystallized under conditions containing magnesium sulfate (0.1–0.3 M) or ammonium sulfate (0.76–1.76 M) and MES buffer (pH 6.0–6.5), both with PEG 3350 as a precipitant.For crystallization, native and selenomethionine-labeled 2.3.et al., 2006XDS , X-ray diffraction data were collected at 100 K on beamline ID23-1 (Nurizzo 2.4.cis–trans isomerization (PPIase) activity was measured with a protease-coupled assay  was diluted in 35 mM HEPES buffer pH 7.6, 150 mM KCl and incubated with 0.1 mg ml−1 bovine α-chymotrypsin for 30 s, in which the pre-existing trans isoform of the substrate peptide was cleaved by chymotrypsin. To start the reaction, CrTIG1 was added in the concentration range 1–25 µM. Absorption at 395 nm was followed in a UV–Vis photometer (Ultraspec 2100 pro). EcTF was used as a positive control and the spontaneous isomerization reaction without trigger factors was used as a negative control. Reaction rates were derived by nonlinear curve fitting to a first-order rate equation using OriginPro and were corrected for spontaneous isomerization.Peptidyl-prolyl 2.5.et al., 2013q-range of 0.025–5 nm−1. Static measurements with three different protein concentrations of CrTIG1 supplemented with 20-meric peptides from Chlamydomonas chloroplast-encoded AtpB (the β subunit of the plastidic ATP synthase) and RbcL (the large subunit of Rubisco) were measured with 1 s exposure times per frame and ten frames per concentration at 293 K. The peptides were first dissolved in DMSO and added to the protein in a tenfold molar excess. Scattering by the corresponding buffer  was measured before and after one run, averaged and subtracted from the protein scattering. As a control, CrTIG1 alone in buffer with the corresponding DMSO concentration was also measured. BSA standards were used to calibrate the I(0) values and the scattering of pure water was used to calibrate the intensity to absolute units  data were collected on the BM29 beamline  and the radius of gyration Rg were evaluated with PRIMUS  the intensity is represented by I(s) = I(0)exp[−(sRg)2/3]. The distance distribution function p(r) and the maximum particle dimension (Dmax) were obtained using GNOM , the mature protein, lacking the N-terminal 67 amino acids of the chloroplast transit peptide, was heterologously expressed in E. coli and purified, yielding protein without remaining affinity tags  and thus did not confirm the truncation of the RBD during crystallization. No putative protease cleavage site by E. coli proteases was predicted between the RBD and the PPIase domain using the ExPASy PeptideCutter tool and MEROPS  and will be referred to here as CrTIG1ΔRBD. Numbering of the amino-acid sequence starts with Ala1 and the secondary-structure elements are numbered according to the full-length EcTF structure . The C-terminal SBD is completely visible except for 15 amino acids at the C-terminus. The middle PPIase domain extends from Phe181 to Leu277. The C-terminal domain contains seven helices and one β-strand and can be divided into a long linker helix (Pro278–Asp332), arm 1 built from three helices (Met333–Leu406) and a second arm  with two helices. A long linker  containing a β-strand followed by an α-helix connects the missing N-terminal domain and the PPIase domain and spans the complete backbone of the protein. The linker is stabilized via a parallel β-sheet built between the β-strand in the linker (β5) and the only β-strand at the C-terminus (β11) .The resulting atomic structure of CrTIG1ΔRBD, while the cradle formed by the PPIase domain and the C-terminal arms is mainly negatively charged . Prominent hydrophobic patches are only found in the cavity of the PPIase domain and in arm 2 of the SBD .Calculation of the surface potential reveals an overall mixed pattern of acidic and basic patches in ed Fig. 1d. PromiCrTIG1ΔRBD with bacterial trigger factors was difficult due to the high flexibility of the domain arrangement, which explains the failure of our approach to solve the structure via molecular replacement based on all available prokaryotic TF structures. The closest structural homologue is the E. coli trigger factor, with a root-mean-square deviation (r.m.s.d.) of between 4.6 and 6.9 Å as determined with DALI . The most similar domain arrangement to that of the PPIase domain of CrTIG1ΔRBD is found in the dimeric structures of EcTF [PDB entries 5owi , consisting of a classical four-stranded antiparallel β-sheet forming a half β-barrel with two inserted 310-helices. This sheet can be extended through a fifth β-strand  at the N-terminus. However, in contrast to the classical FKBP fold, this N-terminal β-strand does not extend the half β-barrel; instead, it builds a separate antiparallel β-sheet together with the C-terminal β-strand 10, which is very unusually elongated . Prominent deviations from other FKBP proteins are further present in the 40s loop between β-strands 7 and 8 and in the 80s loop or so-called ‘flap’ . The 40s loop often divides a β-strand into two parts, but in CrTIG1ΔRBD only the second part of β-strand 8 is present. Instead, a long loop completely differently oriented to EcTF is observed. In the 80s loop, an unusual 310-helix is inserted at the tip of the loop. A second 310-helix is inserted in the 50s loop. β-Strand 7 is extended and is surrounded by an extra-long loop between β-strands 6 and 7, causing a loop crossing. This motif is structurally rare since it is energetically unfavorable .The PPIase domain of ed Fig. 3a. Promip’ Fig. 3c. The 4ps Fig. 3b.CrTIG1ΔRBD towards the arms. This conformation is stabilized via several hydrogen bonds. Two hydrogen bonds are directed towards arm 1 of the C-terminal domain : one formed from the unusual 310-helix in the flap of the PPIase domain and the second one from the neighboring loop after β-strand 7 . Additional stabilizing hydrogen bonds are observed from Lys177 of the linker helix α5 to the prominent loop between β-strands 6 and 7 (to Glu224 and Asp222) and to Gly193 at the end of β-strand 8. Asp222 is also involved in a hydrogen bond to Gln174 in the linker helix .The PPIase domain, which forms the head of the dragon-like structure, is prominently tilted downwards in  7 Fig. 3d. Additix Fig. 3e.EcTF, Glu178, Ile195, Phe198, Tyr221 and Phe233 are conserved and identified as key residues for PPIase activity . The best conserved region between the trigger factors is found in the region surrounding the position of Tyr253 in CrTIG1.Within the PPIase domain of CrTIG1 PPIase domain is the respective domain in the conformation of monomeric E. coli TF  and SlyD . In contrast to CrTIG1, these Arabidopsis PPIases differ in the number of β-strands and in the inlets in the 80s loop. Also, AtFKBP13 and AtFKBP42 contain disulfide bridges  was detected. The performance of the enzyme assay is based on the tetrapeptide assay described previously for EcTF and is also used for determining the activities of chloroplast PPIases . Previously determined kcat/Km values for EcTF with the suc-AFPF-pNA peptide as a substrate were 0.52 mM−1 s−1 . The thus built lap in CrTIG1 mostly contains negative charged amino acids and is mainly surrounded by hydrophobic amino acids . The conformation of the SBD structure most closely resembles the structures of the E. coli trigger factor [PDB entries 1w26 . In CrTIG1 the location of arm 1 is comparable to the respective arm of EcTF, but arm 2 is arranged differently, with a distance of 24 Å and an angle of 27° with monomeric EcTF . Helix α10 in arm 1 is unusually kinked and contains a coiled-coil motif at amino acids 360–392 as predicted by the SMART server .The C-terminal domain of  A Fig. 5e. Helix3.6.CrTIG1ΔRBD might have artificial arrangements caused by truncation of the N-terminal domain, we fitted the X-ray structure together with the rigid-body model of the N-terminal RBD into the SAXS bead model of full-length CrTIG1, which we had published previously , which indicates a good alignment.To determine whether CrTIG1 in solution has a comparable closed conformation with interaction between the PPIase domain and the arms as in the crystal structure.While the N-terminal domain might have more latitude to move in the SAXS envelope, the PPIase domain is likewise tilted down to the SBD and the arms are still in the closed conformation. There seems to be no free space for an open conformation of the PPIase domain and the arms of the SBD. This indicates that full-length CrTIG1 with peptides from the putative interaction partners AtpB (the β subunit of the plastidic ATP synthase) and RbcL (the large subunit of Rubisco) as extracted from peptide-spotting arrays . Taken together, the structure of CrTIG1ΔRBD resembles the bacterial counterparts but displays unique features such as an unusual orientation of the head and arms and also a special surface-charge pattern.We also investigated the conformation of 4.EcTF being by far the best investigated trigger factor and possibly one of the best-understood molecular chaperones in the literature. In contrast, no structural information on chloroplast trigger factors had been obtained until now despite their unique presence in eukaryotic cells and detectable functional deviations from their prokaryotic origin  is AtFKBP20-2, which is involved in the accumulation of the PSII supercomplex in Arabidopsis (Lima et al., 2006However, neither the substitution of the conserved key active-site residues within the hydrophobic pocket of the PPIase domain of 4.2.EcTF exhibits a broad substrate spectrum and seems to bind several hundred nascent polypeptides during their synthesis (Oh et al., 2011in vitro studies indicate less general substrate specificity of CrTIG1 (Rohr et al., 2019EcTF preferably binds peptides with a positive net charge and recognizes aromatic and basic amino-acid residues in peptide substrates (Patzelt et al., 2001CrTIG1 the distribution of the electrostatic and hydrophobic potential shows a mixed pattern on the backbone of the protein, but the inner core between the PPIase domain, the arms and the linker is particularly negatively charged. The amino acids in the peptides identified to bind to both of the eukaryotic trigger factors CrTIG1 and AtTIG1 described in Rohr et al. (2019CrTIG1, the content of nonpolar residues increases to 40% and the ratio of basic/acidic peptides increases to 14%/9%. al. 2019 have no CrTIG1 shows structural homology to SurA. The chaperone activity of SurA in the periplasm of E. coli is essential for outer membrane protein biogenesis (Sklar et al., 2007EcTF showed that β-barrel outer-membrane proteins were the most prominent substrates (Oh et al., 2011CrTIG1 is mainly localized in the stroma and it still remains to be shown whether it contributes to nascent polypeptide maturation of thylakoid membrane proteins, which would explain the similar structural features to SurA.The substrate-binding domain of et al., 2004et al., 2000CrTIG1 remains unclear, but it might be the anchor point or interaction surface for unfolded peptide chains in the active state of the trigger factor. In the inactive state, the coiled-coil motif may stabilize the PPIase domain. It remains to be shown whether formation of the coiled coil may be absent in the ribosome-associated chloroplast TIG1 and thus promote nascent polypeptide binding. Also, for EcTF, MD simulation shows a more compact structure in solution and a framework of intermolecular interactions between the head and arm 1 (Singhal et al., 2013Arm 1 of the SBD forms an unusual coiled-coil motif and also appears to be more flexible than arm 2. Coiled-coil motifs in chaperones have been discussed to mediate the binding of unfolded proteins (Martin 5.C. reinhardtii resembles that of prokaryotic trigger factors, but with different domain orientations as well as an unusual conformation of the arms in the SBD and also uncommon intramolecular domain interactions. While bacterial trigger factors exhibit promiscuous substrate binding and high plasticity, the more distinct negative and hydrophobic patches of CrTIG1ΔRBD in the cradle of the arms and the PPIase domain are compatible with a more specialized function in the chloroplast. Further studies specifically aimed at substrate identification of chloroplast TIG1, such as selective ribosome profiling, are required to precisely understand its role during protein biogenesis. However, the present structure already indicates specific structural adaptation for the binding of proteins in plant organelles. Further, structural availability of the RBD would allow the nature of the ribosome interaction in chloroplasts to be modelled.The crystal structure of a eukaryotic trigger factor is presented. The structure of the trigger factor of the green alga 6.supporting information for this article: Gouet et al. (1999et al. (2019The following references are cited in the  al. 1999 and Made al. 2019.chloroplast trigger factor, 7zgiPDB reference: 10.1107/S2059798322009068/jv5013sup1.pdfSupplementary Figures. DOI:


Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs. The second messenger cyclic guanosine monophosphate (cGMP) regulates a myriad of physiological processes including cellular growth, smooth muscle contractility, cardiovascular homeostasis, inflammation, sensory transduction, bone growth, and neuronal plasticity and learning . In eukaPKGs belong to the AGC family of protein kinases and have N-terminal regulatory (R) and C-terminal catalytic (C) domains. The PKG Iα and Iβ isoforms are splice variants with different N-terminal regions of 89 or 104 residues but the same C-terminal 582 residues that form cyclic nucleotide-binding (CNB) and C-domains . PKG I iG or A  that bind the catalytic cleft, blocking substrate access (Much of our understanding of the mechanism of action of PKGs derives from studies of isolated domains. The R-domain comprises a leucine zipper (LZ) domain, a linker region with an auto-inhibitory (AI) sequence, and two cyclic nucleotide-binding (CNB-A and -B) domains . All thre access . The CNBe access  and are e access . Cyclic e access . A PBC ae access . In PKG e access . Cappinge access .By analogy to PKA, the PKG R-domain is thought to bind via its AI sequence to the C-domain active site to form an auto-inhibited R:C state; binding of cGMP to the R-domain destabilizes this state and allows active kinase to phosphorylate downstream substrates. In contrast to PKG, activation of the PKA R:C state by cAMP dissociates the AI tetramer into separate R and C subunits. In previous work, we showed that a PKG I R-domain comprising two CNB domains but lacking the dimerization domain makes R:R intermonomer contacts through the cGMP bound at CNB-A and at a kink in the interdomain linker between the two CNBs . DisruptHere, we present the first crystal structure of PKG Iβ in which the regulatory domain is free of cyclic nucleotide and adopts an AI state bound to the catalytic domain. The R:C interface reveals specific contacts between the PKG Iβ AI sequence and the active site that help to explain isoform-specific activation constants and the activating effects of phosphorylation in the linker. Comparison with the structures of isolated PKG R- and C-domains shows that the R-domain undergoes local conformational changes and domain rearrangements to assemble a surface that contacts the catalytic core of the C-domain and inhibits its activity. We also present a high-resolution structure of the CNB-A domain with the TAAD mutation, which reveals a closed cGMP pocket. The similarity of this mutant domain to wildtype cGMP-bound structures and its differences with the open pocket in the AI enzyme explain the constitutive activation of the holoenzyme in TAAD. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and activation of PKGs.The asymmetric unit contains two PKG Iβ 71–686 peptide chains see . Structu2+ at the ATP site with the DFG motif adopting an ‘in’ conformation. Both CNB-A and CNB-B PBCs adopt ‘open’ conformations with respect to the β-subdomains, similar to previous CNB domain structures without cyclic nucleotide , K75 (P - 3), and R76 (P - 2), which are critical to auto-inhibition  shows that the longer PKG activation loop is accommodated by small PKG R-domain residues E243 and S246, whereas the PKA activation loop contacts larger PKA RIα residues W260 and L263 at the homologous positions . We infeP - 5) of the RIα subunit, while R133 of the C subunit αD helix (which is homologous to R466 of PKG Iβ) points toward the active site and interacts with E230 of the αF helix.The C-terminal tails of AGC kinases contribute to the overall fold and catalytic function  and are Comparing the AI complex with previous structures of PKG and PKA R-domain fragments bound to cyclic nucleotides  reveals B helix docks to the C-domain , revealing that it adopts a closed conformation that mimics the cGMP-bound wildtype CNB-A . Since tBecause the PBC of the RQ CNB-A domain makes intermonomer crystal contacts, it is possible that these contacts help stabilize the closed state. To determine if the closed state would persist without crystal contacts, we performed molecular dynamics (MD) simulations of the fully solvated domain without nucleotide, and of the wildtype domain with or without nucleotide . MD trajBinding of cyclic nucleotide induces backbone amide Nuclear Magnetic Resonance (NMR) chemical shift perturbations in CNB domains that reflect the degree to which the closed state is populated . To asseWhile the structure in the crystal likely represents a domain-swapped dimer  undergoiWe performed kinase assays under basal conditions (no cGMP) and activating conditions (10 μM cGMP) using Flag-tag purified protein from four separate cotransfections that pair Flag-tagged kinase-dead PKG Iβ (with or without the KR/EE mutation) with untagged kinase-active PKG Iβ (with or without the KR/EE mutation) . With wiKM for VASPtide compared to Kemptide  , and theIn this model of cGMP-mediated PKG activation , the rapPR-cGMPS is a PKG inhibitor that makes many of the same contacts as cGMP with the isolated CNB-B domain, but the PBC loop does not close to contact the phosphorothioate sulfur, and the adjacent helix does not reposition  was cloned into pBlueBacHis2A vector with tobacco etch virus (TEV) protease site before coding sequence. The protein was expressed in SF9 cells with multiplicity of infection of 2 for 48 hr. Cells were suspended in 200 mM potassium phosphate (pH 7.5), 500 mM NaCl (sodium chloride), 1 mM β-mercaptoethanol (Buffer A) and lysed with a cell disruptor . The supernatant was loaded onto HisTrap HP 5 ml column  and eluted with buffer A containing 300 mM imidazole on an ÄKTA purifier system . The His-tag was removed by incubating the eluted protein with TEV protease overnight at 4°C. A second nickel affinity chromatography step was performed to remove TEV protease and flow-through fractions were collected. The tagless protein was further purified by anion exchange chromatography  in 25 mM potassium phosphate (pH 7.5), 1 mM β-mercaptoethanol with and without 1 M NaCl. The Mono Q peak fractions were pooled, concentrated and passed through a Hiload 10/300 Superdex 200 column  equilibrated with 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), 150 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 2 mM MnClE. coli cells . Cells were then grown for an additional 10 hr at 25°C, harvested by centrifugation. Cells were suspended in Buffer A  and lysed with a cell disruptor . The protein was purified with BioRad IMAC resin on a BioRad Profinia purification system and eluted with Buffer A containing 300 mM imidazole. The elution fraction protein sample was incubated with TEV protease at 4°C overnight for His-tag removal. A second nickel affinity chromatography step was performed to remove TEV protease and flow-through fractions were collected. Protein was further purified with gel filtration on a Hi-load 16/60 Superdex-75 column  in 25 mM Trizma (pH 7.5), 150 mM NaCl, and 1 mM TCEP–HCl.A DNA construct containing His-tagged human PKG Iα (79–212) R177Q was cloned into pQTEV and transformed into TP2000 li cells . Cells w−1 by using 30 kDa cutoff Amicon Ultra (Millipore) and initial crystal screening was performed by Mosquito Crystal robot (TTP Labtech) with 100 nl protein drop and 100 nl crystallization solution over 70 µl well. Further optimization was performed with 250 nl protein and 250 nl crystallization solution. Crystals were obtained by vapor diffusion method in 120 mM ethylene glycol, 100 mM Bicine (pH 8.5), 20 % (wt/vol) PEG 8000 at room temperature. The crystals were flash cooled in liquid nitrogen. Diffraction data were collected on Beamline 5.0.1 . To obtain crystals of PKG Iα (79–212) R177Q, 10 mM cGMP  was added to the purified protein, and concentrated to 20 mg ml−1 with a 10 kDa cutoff Amicon Ultra (Millipore). Initial crystal screening was performed by Mosquito Crystal robot (TTP Labtech) with 300 nl protein drop and 300 nl crystallization solution over 70 µl well. Further optimization was performed with 2 µl protein and 2 µl crystallization solution over 500 µl well. Crystals were obtained by vapor diffusion method in 6% Tacsimate (pH 4.5), 18% PEG 3350 at 22°C. The R177Q crystals were dipped in cryoprotectant (Paratone-N) and flash cooled in liquid nitrogen. Diffraction data were collected on Beamline 5.0.1 . Diffraction data for both crystals were processed by using CCP4.iMosflm  protein was concentrated to 20 mg ml.iMosflm  and stru.iMosflm . Coot wa.iMosflm . All the−1 at 20°C was exposed for 3 s at ALS beamline 12.3.1 and small-angle X-ray scattering data were collected over the q range 0.01–0.39 Å−1. Primary data reduction and processing were performed with the ScÅtter pipeline and ATSAS suite. The fitted values for I(0) and Rg from Guinier analysis and P(r) analysis agree closely see I(0) is consistent with a monomer and the experimental Rg is consistent with the cis-conformation. Ab initio analysis was performed with DAMMIF/DAMMIN, computation of model intensities was performed with the FoXS webserver, and crystal structures were fit into models using SUPCOMB.PKG Iβ (71–686) protein at 3.5 mg mlhPKG Iα and pFLAG hPKG Iβ  and two times Tris buffer . Elution was performed using a Tris buffer containing 2.5 mM desthiobiotin. Subsequently, a buffer exchange with Tris buffer was performed to store the proteins without the desthiobiotin to avoid possible interference with downstream analyses.FLAG-TwinStrep-tagged hPKG Iα and Iβ were generated by cloning the respective DNA sequence from pFLAG  hPKG Iβ  using 5′ plasmid . The genKact) and Michaelis–Menten constants (KM) were determined using an enzyme-coupled spectrophotometric kinase assay as described by 2, 1 mM ATP, 1 mM phosphoenolpyruvate, 15.1 U/ml lactate dehydrogenase, 8.4 U/ml pyruvate kinase, 230 µM 1,4-Dihydronicotinamide adenine dinucleotide (NADH), 5 mM β-mercaptoethanol) was mixed with the hPKG samples in a glass cuvette and the reaction was started by adding the respective substrate peptide  to a final concentration of 1 mM. For basal activities, measurements were performed without cGMP. Maximal kinase activity was determined in the presence of a final cGMP concentration of 200 µM. The absorption at 340 nm was monitored for 30–120 s using a double beam photometer  and the slope was determined to calculate the specific kinase activity.Specific kinase activities, activation constants (Kact) was determined by mixing assay mix with a kinase mix and a dilution series of cGMP in a 384-well plate  in a ratio of 2:1:1 and monitoring the absorption at 340 nm for 200–300 s in a microplate reader . Protein activity was calculated according to the Lambert–Beer law and converted to the observed catalytic activity at the given substrate concentration of 1 mM (kobs). Values of kobs were plotted against the cGMP concentration on a logarithmic scale and Kact was determined by fitting the data using a sigmoidal dose–response fit with a variable slope. The activation constant is defined as the cGMP concentration at which half-maximal kinase activity occurs.The activation constant (KM). Here, assay mix, kinase mix with cGMP  and a dilution of VASPtide or Kemptide, respectively, were mixed in a 384-well microplate in a 2:1:1 ratio and processed as described. Calculated kinase activities were plotted against the substrate concentration and fitted to the Michaelis–Menten equation to obtain KM and vmax.A similar procedure was followed for the determination of Michaelis–Menten constants  D2O, phosphodiesterase (PDE), 1 mM ATP, and 10 mM MgCl2. A cGMP-bound sample was prepared similarly, with the addition of 1 mM cGMP to the apo protein solution. All HSQC spectra were recorded with 16 scans and 1-s recycle delay. The spectra included 128 (t1) and 2048 (t2) complex points with spectral widths of 38.0 and 16.2 ppm for the 15N and 1H dimensions, respectively. Carrier frequencies of 1H and 15N were set at the water and central amide region, respectively. All spectra were acquired at 300 K with a Bruker Avance 700 MHz NMR spectrometer equipped with a 5 mm TCI cryoprobe. The spectra were processed with TOPSPIN and analyzed using Sparky  reported . NMR samg Sparky .All MD simulations were performed using the NAMD 2.12 software  on the S This crystal structure of nearly full-length human cGMP-dependent protein kinase Iβ (PKG Iβ) provides convincing new insights into how in the absence of cGMP the activity of the catalytic domain is held in check by intramolecular interactions between both the upstream regulatory cGMP-binding domains and autoinhibitory segment and the catalytic domain, and how cGMP binding to the two cGMP-binding domains can relieve these inhibitory constraints leading to an increase in catalytic activity. The regulatory interactions in PKG Iβreveal similarities and differences to the way in which the cAMP-dependent protein kinase (PKA) regulatory domain inhibits the PKA catalytic subunit. The new structure of the activating PKG Iα R177Q CNB-A domain mutant, which resembles a cGMP-bound wild-type CNB-A domain, provides a nice explanation for how this point mutation activates PKG Iα and leads to the development of the TAAD (Thoracic Aortic Aneurysms and Dissections) syndrome. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Tony Hunter as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ping Zhang (Reviewer #2).Thank you for submitting your article "An auto-inhibited state of protein kinase G and implications for selective activation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.P. falciparum PKG structure (doi: 10.1073/pnas.1905558116) would be informative. Some additional specific suggestions for improvement can be found in the individual reviews. We look forward to receiving a revised version.The three reviewers are in general agreement that your nearly-full-length human PKG1β structure is a significant advance in our understanding of the regulation of PKG by cGMP. No new experimental analysis is essential, although additional structure-function validation would strengthen your conclusions. However, all the reviewers agree that the paper is too long and difficult to read, and needs to be shortened and rewritten so that the main conclusions about how activity is held in check in the absence of cGMP, how cGMP binding activates PKG, and the extent to which the regulation of PKG is similar to and differs from that of the PKA C subunit by the R subunit and cAMP are more clearly articulated in each section. In addition, it would be useful to include some further discussion of why PKG is a dimer and whether there is functional crosstalk between the two monomers in a dimer, if negative regulation of kinase activity can be achieved through purely intramolecular interactions. In addition, a comparison of the human PGK1β structure with that of the full length Essential revisions:The main essential revision is to undertake a major rewrite of the paper to shorten it and make clear the main conclusions in each section regarding the mechanisms of negative regulation of PKG kinase activity in the absence of cGMP and how it is activated upon binding cGMP, and how these mechanisms are similar to and differ from the regulation of the PKA C subunit by the R subunit and cAMP.Reviewer #1 (Recommendations for the authors):This paper is very long and was a hard slog to read through, and there may be a very limited number of people who will take the trouble to do this. The detailed discussion often obscures the main take home conclusion in each section. When the paper is rewritten and shortened to make the main conclusions clearer, perhaps some of the key interactions could be summarized in a graphic table instead of being described in detail in the text.The functional consequences on PKG Iβ kinase activity and cGMP regulation of some additional mutations in key residues proposed to be involved in autoinhibition would strengthen the authors conclusions.Reviewer #2 (Recommendations for the authors):eLife, once the following comments are addressed.The manuscript will be very well-suited for publication in – The cooperative nature of cGMP binding is mentioned in the introduction (line 58), however in the model , it is not clear of cGMP binding is cooperative only within a R/C dimer, or with two dimers in a full length R2/C2 tetramer. One approach would be to determine Hill coefficients to establish if the binding and activation by cGMP is cooperative in the tetramer.It would be interesting to explain and develop further why PKG exists as a R-C:R-C homodimer and whether there is crosstalk between the two R-C monomers. One issue that is not addressed by the manuscript explicitly is the cooperativity of cGMP binding in full-length PKGIβ homodimer. The cooperative nature of cGMP binding is mentioned in the introduction (line 58), however in the model , it is not clear of cGMP binding is cooperative only within a R-C monomer, or with two R-C monomers in a full length R-C:R-C dimer. One approach would be to determine Hill coefficients to establish if the binding and activation by cGMP is cooperative in the R-C:R-C dimerOne general comment is that the manuscript is very long . Please consider streamlining the manuscript – you may consider condensing some of the discussion of the interfaces and moving parts of it to a supplementary material section or table.Reviewer #3 (Recommendations for the authors):eLife. Suggestions for improving presentation:I have no technical/experimental concerns with the work, it appears to be technically sound on all fronts and is an important advance suitable for publication in 1. I feel that contrast/compare with PKA is given short shrift. I want to know what is the same and what is different, and it is not easy to get that at a glance. That it looks almost the same as PKA (if indeed it does) does not make the present structure less interesting. Perhaps we could learn this right from the start, in the "overall structure" section?2. The authors frequently "bury the lede" as they say in the newspaper trade. For example, the overall structure section starts out talking about individual domains and how they differ from previous structures of isolated domains. Actually, I am not sure they ever get around to describing the overall structure in this section, but there is a picture of it in Figure 1. Why not start with something like: In the autoinhibited state, the cyclic-nucleotide binding domains bind on either side of the kinase C-lobe, positioning the autoinhibitory segment to bind as a pseudosubstrate at the kinase active site. This overall organization is  that of protein kinase A….3. In figure 1b, it is hard to get a sense of the structure of the kinase domain, and where on the kinase domain the regulatory domain is binding. At a minimum, labeling N- and C-lobes would help. Or perhaps a ribbon representation of the kinase would be better? Maybe with semi-transparent surface?4. Would also be nice to get the comparison with PKA into Figure 1. . Certainly up to the authors to decide what to describe/include in their manuscript, but I think it would be interesting and helpful to have a comparison of R:C contacts of PKA vs PKG, perhaps in the form of a sequence alignment with symbols to represent the various interdomain interfaces. What is conserved and what is unique to PKG?5. Is there experimental evidence  for dimerization of the CNB domain module in the nucleotide-bound state? If not, the text/figures describing the reorientation of these domains between autoinhibited and active (dimerized) states feels over-done. Essential revisions:The main essential revision is to undertake a major rewrite of the paper to shorten it and make clear the main conclusions in each section regarding the mechanisms of negative regulation of PKG kinase activity in the absence of cGMP and how it is activated upon binding cGMP, and how these mechanisms are similar to and differ from the regulation of the PKA C subunit by the R subunit and cAMP.We have significantly shortened the main text and attempted to state the regulation and activation mechanisms mediated by dynamic R-C interactions. Our main text includes now 6774 words (~35000 characters) reduced by ~1000 words  from the original submission.P. falciparum PKG structure at the overall structure section and provided RMSD values.We addressed why PKG is a dimer and its role in the cooperativity in the Discussion. We also provided our thoughts on functional crosstalk between the two monomers in a dimer in the same section. We also compared our PKG Iβ structure with the PKA R:C heterodimer structure and the full-length Reviewer #1 (Recommendations for the authors):This paper is very long and was a hard slog to read through, and there may be a very limited number of people who will take the trouble to do this. The detailed discussion often obscures the main take home conclusion in each section. When the paper is rewritten and shortened to make the main conclusions clearer, perhaps some of the key interactions could be summarized in a graphic table instead of being described in detail in the text.We thank the reviewer for encouraging us to focus on the most important findings. We have relegated many of the details about R:C contacts to Supplementary file 3, which can be compared directly with Figure 3 of Kim et al., 2005, Science to appreciate similarities and differences between PKG and PKA auto-inhibition.The functional consequences on PKG Iβ kinase activity and cGMP regulation of some additional mutations in key residues proposed to be involved in autoinhibition would strengthen the authors conclusions.Our structure enabled us to design the KR/EE mutation and to provide structural interpretations for many mutational analyses performed by others over the last two decades, which we cite and discuss.Reviewer #2 (Recommendations for the authors):The manuscript will be very well-suited for publication in eLife, once the following comments are addressed.– The cooperative nature of cGMP binding is mentioned in the introduction (line 58), however in the model , it is not clear of cGMP binding is cooperative only within a R/C dimer, or with two dimers in a full length R2/C2 tetramer. One approach would be to determine Hill coefficients to establish if the binding and activation by cGMP is cooperative in the tetramer.It would be interesting to explain and develop further why PKG exists as a R-C:R-C homodimer and whether there is crosstalk between the two R-C monomers.We addressed the cooperativity of cGMP binding/activation of PKG Iβ by citing Smith et al., and stating that “auto-inhibited monomeric PKG Iβ leucine zipper deletion mutants are activated by cGMP without exhibiting cooperativity, so dimerization is dispensable for auto-inhibition but essential for cooperative activation .” We further propose our thoughts on why PKG exists as a R-C:R-C homodimer by stating “If each monomer of a leucine zipper-mediated dimer has two bound cGMP, the PBC-A and bent interdomain helices of one monomer can associate with the same regions of the other monomer to which it is tethered and assemble into an antiparallel R:R interface  . The formation of this interface further stabilizes the activated state . This assembly helps sustain kinase activation since it occupies much of the R-domain surface that would otherwise be free to associate with the C-domain; it also shields the bound cGMP from solvent, which reduces the rate at which it might be released and degraded by phosphodiesterases .”One issue that is not addressed by the manuscript explicitly is the cooperativity of cGMP binding in full-length PKGIβ homodimer. The cooperative nature of cGMP binding is mentioned in the introduction (line 58), however in the model , it is not clear of cGMP binding is cooperative only within a R-C monomer, or with two R-C monomers in a full length R-C:R-C dimer. One approach would be to determine Hill coefficients to establish if the binding and activation by cGMP is cooperative in the R-C:R-C dimerThe cooperativity of cGMP activation for the full-length dimer is reported in Smith et al., and is cited in the revised manuscript, as is the lack of cooperativity for constructs lacking the LZ. We use these reported cooperativity data and our data showing the lack of inter-monomer effects on auto-inhibition to argue that cGMP cooperativity derives from R:R interactions in the activated state .This point was addressed in the responses to comments from reviewer 1.Reviewer #3 (Recommendations for the authors):I have no technical/experimental concerns with the work, it appears to be technically sound on all fronts and is an important advance suitable for publication in eLife. Suggestions for improving presentation:1. I feel that contrast/compare with PKA is given short shrift. I want to know what is the same and what is different, and it is not easy to get that at a glance. That it looks almost the same as PKA (if indeed it does) does not make the present structure less interesting. Perhaps we could learn this right from the start, in the "overall structure" section?We added sentences describing its similarity to PKA at the overall structure section as suggested .2. The authors frequently "bury the lede" as they say in the newspaper trade. For example, the overall structure section starts out talking about individual domains and how they differ from previous structures of isolated domains. Actually, I am not sure they ever get around to describing the overall structure in this section, but there is a picture of it in Figure 1. Why not start with something like: In the autoinhibited state, the cyclic-nucleotide binding domains bind on either side of the kinase C-lobe, positioning the autoinhibitory segment to bind as a pseudosubstrate at the kinase active site. This overall organization is  that of protein kinase A….This was addressed in the previous response.3. In figure 1b, it is hard to get a sense of the structure of the kinase domain, and where on the kinase domain the regulatory domain is binding. At a minimum, labeling N- and C-lobes would help. Or perhaps a ribbon representation of the kinase would be better? Maybe with semi-transparent surface?We modified Figure 1b so that a ribbon representation of the kinase is visible with semi-transparent surface. We also added labels for small (N) and large (C) lobes of the kinase domain, either termini, and the ordered residues at the R-C linker.4. Would also be nice to get the comparison with PKA into Figure 1. . Certainly up to the authors to decide what to describe/include in their manuscript, but I think it would be interesting and helpful to have a comparison of R:C contacts of PKA vs PKG, perhaps in the form of a sequence alignment with symbols to represent the various interdomain interfaces. What is conserved and what is unique to PKG?We added sentences describing how R:C contacts in PKA are compared to PKG in overall structure section , the R:C interface section, and the C-domain architecture and comparison with PKA section. We also emphasized how these differences might play important roles in minimizing cross-talk between the cyclic nucleotide signaling pathways in line 234, pg 9. We generated a R-C interaction table (Supplementary file 3), which can be compared directly with Figure 3 of Kim et al., 2005, Science to appreciate similarities and differences between PKG and PKA contacts that mediate auto-inhibition.5. Is there experimental evidence  for dimerization of the CNB domain module in the nucleotide-bound state? If not, the text/figures describing the reorientation of these domains between autoinhibited and active (dimerized) states feels over-done.Discussing this structure is important because it provides a possible physical basis for cooperativity of cGMP binding; literature shows that the LZ is needed for cooperativity, and our data disprove the possibility that intermonomer interactions contribute to the auto-inhibited state.


Neurodegenerative diseases are a set of progressive and currently incurable diseases that are primarily caused by neuron degeneration. Neurodegenerative diseases often lead to cognitive impairment and dyskinesias. It is now well recognized that molecular events precede the onset of clinical symptoms by years. Over the past decade, intensive research attempts have been aimed at the early diagnosis of these diseases. Recently, exosomes have been shown to play a pivotal role in the occurrence and progression of many diseases including cancer and neurodegenerative diseases. Additionally, because exosomes can cross the blood–brain barrier, they may serve as a diagnostic tool for neural dysfunction. In this review, we detail the mechanisms and current challenges of these diseases, briefly review the role of exosomes in the progression of neurodegenerative diseases, and propose a novel strategy based on salivary neuronal exosomes and nanoparticle tracking analysis that could be employed for screening the early onset of neurodegenerative diseases. Timely diagnosis of neurodegenerative diseases at an early stage provides an effective means to manage the ‘impending burden’ of the aging population globally. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases. In terms of prevalence, AD tops the list, followed by PD. The other spectrum of neurodegenerative diseases encompasses frontotemporal dementia (FTD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS) , 2. AccuSNCA, parkin, DJ-1, PINK-1 (PTEN-induced kinase 1), and Leucine-rich repeat kinase 2 (LRRK2) [On the other hand, PD is a chronic neurodegenerative disease characterized by motor impairments due to the death of dopaminergic neurons within the substantia nigra. Cognitive impairments can also follow during the course of the disease . General (LRRK2) . Althoug (LRRK2) –13.Both of these neurodegenerative diseases are currently incurable, and the pathological events start a decade before symptoms become severe enough to be diagnosed clinically using the current criteria. The development of PD has three stages, preclinical, prodromal, and clinical, which are characterized by the onset of neurodegeneration in the absence of symptoms, the presence of non-motor symptoms, and the occurrence of motor symptoms, respectively , 15. TheALS is a neuromuscular disorder resulting from protein inclusions formed from TAR DNA-binding protein of 43 kDa (TDP-43), Cu/Zn superoxide dismutase (SOD-1) or fused in sarcoma (FUS) within motor neurons , which rIT15 gene that results in abnormal numbers of glutamine repeats (polyQ) in the huntingtin (Htt) protein. Six to 34 CAG repeats are normal but a person with over 40 repeats will develop HD and usually die 10–15 years after disease onset [HD is also an incurable neurodegenerative disease and the most common form of inherited neurodegenerative disease. HD is characterized by uncontrolled and excessive motor movements along with a significant cognitive loss. HD is associated with abnormal expansion of a CAG repeat in the se onset . Therefose onset , 27.With a need for an efficient diagnostic methodology for neurodegenerative diseases, a variety of approaches have been tried ranging from tracking subtle functional changes in the brain using positron emission tomography (PET) –30 to idExosomes are tiny extracellular vesicles ranging from 30 to 100 nm in diameter, and are released by all cell types. These vesicles are formed from late endosomes via inward budding of multivesicular bodies (MVBs). Invagination of endosomal membranes results in the formation of intraluminal vesicles (ILVs) within large MVBs Fig. . During Extracellular vesicles including exosomes can be isolated by several techniques such as differential ultracentrifugation, size exclusion chromatography, ultrafiltration, immunoaffinity capture, and polyethylene glycol-based precipitation. Efficient isolation of exosomes from different biofluids has been largely studied and nicely reviewed elsewhere –62, thusAnother important class of molecules contained within exosomes are miRNAs, which are usually small, non-coding, and about 22 nucleotides in length. A miRNA can repress the translation or regulate the degradation of over 100 mRNAs and one mRNA may be regulated by multiple miRNAs. Thus, they form a powerful gene regulation network and are involved in key biological processes including cell signaling, apoptosis, neuronal development, and plasticity , 70. miRFurthermore, exosomes have been strongly linked to the pathogenesis and progression of many neurodegenerative diseases , 76. In Some exosomal miRNAs have also been reported as PD biomarkers. For instance, miR-19b, miR-24 and miR-195 can be used for PD diagnosis, based on the target scan tool. Cao et al.  related 1−42, and p-S396-tau [Another set of misfolded proteins including superoxide dismutase 1 and TDP-43, which have shown associations with ALS, have been identified in exosomes , 85. AlsS396-tau –88. AddiS396-tau . AnotherS396-tau .Several studies have pointed out that exosomes can also transport the expanded polyglutamine tract of both Htt RNA and protein as well as the mutant huntingtin protein (mHtt) aggregates, and thus trigger HD-related behavioral and pathological features –92. Htt GRN) [C9orf72 (chromosome 9 open reading frame 72) [MAPT (microtubule associated tau) [GRN result in the reduction of exosomes and the alteration of their composition [18F-FDG-PET [FTD is considered as a second most common cause of dementia with an age of onset < 65. In FTD, the most common mutations are from three genes: granulin (GRN) , C9orf72rame 72)  and MAPTted tau) . It has position . Neurofiposition , 99. Ano-FDG-PET .Recently, the biomarker potential of salivary neuronal exosomes has been explored in neurodegenerative diseases. Saliva is a type of easily accessible biofluid and isolation of salivary exosomes is a non-invasive, painless, and relatively simple procedure when compared with blood and CSF sampling . Some reOn the other hand, some research findings indicate that the production of exosomes may be involved in the improvement of pathological phenotype of diseases like AD , 107 andk = Boltzmann constant, T = temperature, and η = solvent viscosity. The particle size is reported as the hydrodynamic radius, Rh, determined by following the 2D trajectory of each particle over a tracking time t.NTA has emerged as a state-of-the-art method for the characterization of exosomes , 113. ThThe advantage of NTA is that individual particles are tracked, hence one can obtain both the concentration and the size distribution, rather than just a mean size. One disadvantage with NTA is that smaller particles are not easily measurable, as the minimum size for detection depends on the refractive index increment. In addition, particles larger than 1 μm diffuse slowly and thus are also difficult to measure with NTA . A seemiThe aggregation of proteins is central to many neurodegenerative disorders. The NTA system has been used for the characterization of fibrillar protein aggregates including Aβ and polyglutamine peptides , and anaDespite significant advances in clinical imaging technologies, there exist several unaddressed challenges in the accurate diagnosis of neurodegenerative diseases. As the molecular changes in these diseases begin as early as 10–20 years prior to the clinical manifestations, tracking molecular events could aid in the early diagnosis. In this regard, exosomes are considered as new “hotspots” since their role in the progression of various diseases is turning out to be quite critical. Through this article, we put forward a screening technology, which could be employed along with other already established subjective assessments and imaging methods for early diagnosis of neurodegenerative disorders. Fluorescence NTA has the potential to quantitate neuronally derived exosomes from diseased and healthy cohorts, based on their specific surface markers.As of now, there are several studies elucidating concentration differences of exosomal particles between diseased and healthy volunteers. But cautiously, more studies with increased sample sizes are needed to validate this methodology and  make it a “mass-screening technology” in the future. Another necessity of particular interest is the follow-up of participants in such studies to gain more confidence in such a screening methodology. In the foreseeable future, more efforts should be made in similar areas to tackle the increasing burden of neurodegenerative diseases, in addition to drug-based efforts for curing these debilitating diseases.


Hemerythrin is an oxygen-binding protein originally found in certain marine invertebrates. Oxygen reversibly binds at its non-heme diiron center, which consists of two oxo-bridged iron atoms bound to a characteristic conserved set of five His residues, one Glu residue, and one Asp residue. It was recently discovered that several bacteria utilize hemerythrin as an oxygen- and redox-sensing domain in responding to changes in cellular oxygen concentration or redox status, and immediately adapt to these environmental changes in order to maintain important physiological processes, including chemotaxis and c-di-GMP synthesis and degradation. This Mini Review focuses on the recent progress made on structural and functional aspects of these emerging bacterial hemerythrin domain-containing oxygen and redox sensors, revealing characteristic features of this family of proteins. Bacteria sense various environmental changes to survive and respond immediately in order to maintain cellular homeostasis. To this end, bacteria utilize many sensor proteins that have evolved for sensing various specific environmental stimuli. In particular, oxygen and redox sensor proteins often contain a redox-active metal-containing prosthetic group such as a heme, non-heme iron or iron-sulfur cluster for sensing intracellular and extracellular oxygen concentrations and redox status.In general, sensor proteins are composed of an N-terminal cofactor-containing sensing domain and C-terminal functional domain. Association/dissociation of oxygen molecules with/from the metal center (in the case of an oxygen sensor) or reduction/oxidation of the metal center (in the case of a redox sensor) induces structural changes in the sensing domain. These structural changes are then transmitted to the functional domain, thereby switching on/off transcription or catalytic reactions .Phascolopsis gouldii , is the best studied  domain, MCP domain and hemerythrin domain  and a rate constant of 160 s−1 for the corresponding oxygen dissociation (koff) have been measured, with a subsequent dissociation constant (Kd) of 0.3 µM  (2 ligand to the non-heme diiron site. The autooxidation of DcrH (t1/2 = 22 min) has been measured to be 54 time faster than that of P. gouldii hemerythrin (t1/2 = 20 h)  . This hi = 20 h) —indicatiR, was found to be stable for over 1 week  was observed to be bound at the position that oxygen would normally bind.Crystal structures of diferric (met), azido-diferric (azidomet), and diferrous (deoxy) complexes of the isolated hemerythrin domain of DcrH were determined to resolutions of 2.1, 1.5, and 2.2 Å, respectively . These cectively . The oveectively . These sectively . The GluR structures, suggesting that the structural change in the loop region is related to reduction of the semimetR form to the deoxy form  binding not inducing conformational change . Furtheroxy form . Thus, Doxy form . However2-binding protein to that of an O2-activating enzyme have also been made -cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous intracellular second messenger that regulates cell motility, biofilm formation, and other processes in bacteria . IntraceVibrio cholerae Bhr-DGC is the first demonstrated example of an enzymatically active full-length protein containing a bacterial hemerythrin domain and showing redox-dependent catalysis (−1) of the diferrous (FeIIFeII) form was measured to be 10 times higher than that of the diferric (FeIIIFeIII) form (0.073 min−1), demonstrating a redox-dependent catalysis. The air-reoxidized protein was observed to revert to the lower activity, indicative of the reversibility of this redox interconversion. The azido-diferric form is also inactive. Thus, the catalytic activity is regulated by redox change, but not ligand binding. In addition, overexpression of Bhr-DGC in Escherichia coli cells was found to induce biofilm formation. Overall, Bhr-DGC catalyzes the formation of c-di-GMP from GTP to regulate biofilm formation in the low-oxygen environment in V. cholerae.atalysis . Bhr-DGCatalysis . AlthougFerrovum sp. PN-J185, as the second example of a full-length protein containing a bacterial hemerythrin domain (−1), whereas no activity was detected for the oxidized ferric iron form. The azide-bound form was also observed to be inactive. This trend of redox-regulated, but not ligand-regulated, catalytic activity is similar to that of Bhr-DGC. This redox interconversion was observed to be reversible, suggesting that Bhr-HD-GYP is important for redox sensing in vivo. The currently available data suggest that Bhr-HD-GYP regulates the switch between the motile planktonic and sedentary biofilm-associated lifestyles in response to changes in cellular redox status or oxygen concentration.Recently, Bhr-HD-GYP was discovered from the iron-oxidizing Gram-negative bacterium n domain . This prn domain , and wasn domain . Non-hemn domain . HoweverNote that Bhr-HD-GYP is currently the only known bacterial hemerythrin domain-containing sensor phosphodiesterase. No other type of phosphodiesterase containing an EAL domain has yet been discovered.1B-type ATPase is a member of a family of integral membrane proteins that use the energy of ATP hydrolysis to transport transition metal ions across cell membranes, and is named after the formation of a phosphorylated intermediate during catalysis. Some P1B-5-ATPases (proteins constituting the fifth member of P1B-ATPase) contain each a C-terminal hemerythrin-like domain  conserved iron ligand residues, 2) fusion with an N- or C-terminal functional domain in a single polypeptide, 3) larger substrate tunnel, 4) relatively rapid autooxidation, and 5) oxygen-binding and/or redox-dependent catalysis .Bacterial hemerythrin domain-containing sensors exhibit considerable similarities to marine invertebrate hemerythrins, but there are also significant differences. Except for DcrH, the oxy state in this type of sensor protein is generally unstable, unlike the case for invertebrate hemerythrins, as no oxygen adduct has been observed for Bhr-DGC and Bhr-HD-GYP. Autoxidation of these bacterial hemerythrin sensors, including DcrH, Bhr-DGC, and Bhr-HD-GYP, occurs much more rapidly than does that of invertebrate hemerythrins—and this feature at least in the case of DcrH is apparently due to its large observed substrate tunnel, which facilitates rapid oxygen binding and solvent access to the oxygen-binding pocket . This tuII heme in the sensor domain regulates its catalytic activity  between active and inactive states of the full-length protein is required, and is the next challenge to be addressed.Pseudomonas aeruginosa (In addition to bacterial hemerythrin domain-containing sensors, single-domain hemerythrins from bacteria have been identified and characterized recently, for example, microoxic hemerythrin from ruginosa . Furtherruginosa  have alsFinally, it should be noted that a search of the genomic database shows a bacterial hemerythrin domain in a wide range of bacteria , where i


In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents. The virus is the seventh coronavirus that has infected humans and the third β-coronavirus to cause an outbreak in the 21st century, following the Severe Acute Respiratory Syndrome (SARS-CoV) of 2003 and the Middle East Respiratory Syndrome (MERS) of 2012 [Severe acute respiratory syndrome SARS-CoV-2), which emerged in Wuhan, China in December 2019, has now spread worldwide [, which e of 2012 ,5. A wid of 2012 ,7,8. The of 2012 . To date of 2012 ,12,13.Several X-ray crystal structures of SARS-CoV-2 main protein enzymes (3CLpro or Mpro) combined with lead compounds serving as inhibitors have recently been announced ,15,16. TThough X-ray crystal structures of the entire enzyme have been published, some of structural and catalytic roles of domain III are still unclear. For example, since the dimeric interfaces of domain III in the crystal structure is quite narrow, its role in dimerization seems limited. In addition, its contribution in the catalytic mechanism is also not well explained. In this study, we determined the X-ray crystal structure of the catalytic domain of SARS-CoV-2 3CLpro and performed fluorescence resonance energy transfer (FRET)-based enzymatic assays against entire and catalytic domains. A new insight for the catalytic domain is discussed in this study.The crystal structure of the catalytic domain of SARS-CoV-2 CLpro was determined at 2.27 Å resolution. In the final model, 182 residues were included. The first six residues (Ser1 and Met6) and the last eight residues (Gln189 to Thr196) in the LH-loop were not visible in the final model though they were predicted to form a short β-strand in PSIPRED . The oveIn order to find the oligomeric state of the catalytic domain in solution, a gel chromatographic experiment was performed. Unexpectedly, only a monomeric form was detected . This im191AQAAGT196 sequence was compensated by the salt-bridge between Arg131 and Asp197. As a result, the entire SARS-CoV-2 3CLpro may exert a full enzyme activity. In addition, Gln189 on the LH-loop takes part in binding with chemicals, as shown in several X-ray crystal structures [In the X-ray crystal structure of the catalytic domain, the eight residues included in the LH-loop are not visible in this construct. In contrast, the LH-loop of the entire SARS-CoV-2 3CLpro is well defined due to the following interactions. First, Gln192 interacts with the backbone oxygen and nitrogen atoms of Val186 . Second,ructures ,34. Therm and Vmax values. When 1 µM 3CLpro was mixed with various concentrations of FRET substrate (0–30 µM), the initial velocity was measured and plotted against the substrate concentration. The curve fitting the Michaelis–Menten equation gave the best-fit values of Km and Vmax as 5.723 µM and 566.137 RFU/s for the entire domain and as 6.152 µM and 71.135 RFU/s for the catalytic domain. The calculated kcat/Km were 712.247 s−1 M−1 and 83.253 s−1 M−1, respectively  for protein expression. E. coli BL21 (DE3) cells were grown on Luria–Bertani (LB) agar plates containing 150 μg ml−1 ampicillin. Several colonies were picked and grown in capped test tubes with 10 mL LB broth containing 150 μg ml−1 ampicillin. A cell stock composed of 0.85 mL culture and 0.15 mL glycerol was prepared and frozen at 193 K for use in a large culture. The frozen cell stock was grown in 5 mL LB medium and diluted into 1000 mL fresh LB medium. The culture was incubated at 310 K with shaking until an OD600 of 0.6–0.8 was reached. At this point, the expressions of SARS-CoV-2 3CLpro and its catalytic domain were induced using isopropyl-β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. The culture was further grown at 310 K for 3 h in a shaking incubator. Cells were harvested by centrifugation at 7650 g (6500 rev min−1) for 10 min in a high-speed refrigerated centrifuge at 277 K. The cultured cell paste was resuspended in 25 mL of a buffer consisting of 20 mM Tris pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 μg ml−1 DNase I. The cell suspension was disrupted using an ultrasonic cell disruptor . Cell debris was pelleted by centrifugation at 24,900× g  for 30 min in a high-speed refrigerated ultra-centrifuge at 277 K.The coding sequence of SARS-CoV-2 3C-like proteinase (NCBI Ref. seq. YP_009725301.1) was synthesized chemically by Bioneer, Daejeon, Korea and cloned into a bacteriophage T7-based expression vector. Its catalytic domain was synthesized from M1 to T196 of 3CLpro. The catalytic one was synthesized by adding ENLYFQGGG (TEV protease cleavage site) and the entire enzyme by adding AVLQ (autocleavage site) at the N-terminus. The plasmid DNA was transformed into The entire SARS-CoV-2 3CLpro was purified by affinity chromatography using a 5 mL Hi-Trap His column . The column was equilibrated with a buffer consisting of 20 mM Tris pH 7.5, and the pooled fractions were loaded. The fractions of 3CLpro-His tag were mixed with Human Rhinovirus (HRV) 3C protease-His-tag at a molar ratio of 10:1 to remove the C-terminal His tag. The His-tag cleaved 3CLpro was further purified by affinity chromatography using a 5 mL Hi-Trap His column  and the flow-through was collected. The purified protein was buffer exchanged into 20 mM Bis-Tris pH 7.5 using Vivaspin 20 MWCO 10 kDa , a centrifugal device. SDS–PAGE showed one band around 34 kDa , corresponding to the molecular weight of SARS-CoV-2 3CLpro.The catalytic domain of SARS-CoV-2 3CLpro was purified by affinity chromatography using a 5 mL Hi-Trap His column . The column was equilibrated with a buffer consisting of 20 mM Tris pH 7.5, and the pooled fractions were loaded. The fractions of 3CLpro-His tag were mixed with TEV protease-His-tag at a molar ratio of 10:1 to remove the N-terminal His tag. The His-tag cleaved 3CLpro was further purified by affinity chromatography using a 5 mL Hi-Trap His column , and the flow-through was collected. The purified protein was buffer exchanged into 20 mM Bis-Tris pH 7.5 using Vivaspin 20 MWCO 10 kDa , a centrifugal device. SDS–PAGE showed one band around 22 kDa , corresponding to the molecular weight of the catalytic domain of SARS-CoV-2 3CLpro.excitation = 340 nm and λemission = 490 nm, with bandwidths = 9 and 15 nm, respectively) of EDANS upon peptide hydrolysis as a function of time. The assays were conducted in black, 96-well plates (Nunc) in 300 μL assay buffers containing proteases and substrates, as follows: For the measurements of Km/Vmax, a proteolytic reaction with 2 µM 3CLpro in 300 µL of the reaction buffer was carried out at 310 K in a SpectraMax i3x (Molecular Devices) with filters for excitation at 360/40 nm and emission at 460/40 nm. The reaction time was 2 h for both of the SARS-CoV-2 3CLpros at 310 K. A FRET substrate concentration ranging from 0 to 30 µM was applied. The initial velocity was plotted against the FRET concentration with the classic Michaelis–Menten equation in GraphPad Prism 7.03 . All reactions were carried out in triplicate.The custom-synthesized fluorogenic substrate, DABCYL-KTSAVLQSGFRKME-EDANS , was used as a substrate for the proteolytic assay using the SARS-CoV-2 3CLpro . This su−1, fractions of 0.3 mL were collected, and 10 mg mL−1 of catalytic domain and 5 mg mg mL−1 of the entire domain of SARS-CoV-2 3CLpro were loaded and then eluted with a buffer consisting of 25 mM sodium phosphate (pH 7.0) or 20 mM Tris (pH 7.8) and 100–300 mM NaCl. The column calibration was conducted with 2 proteins: human albumin (65 kDa) and TCTP (34 kDa).Size-exclusion chromatography experiments were performed using a 10/30 Superdex 200 column  connected to an ÄKTA-explorer system. The typical flow rate was 0.4 mL minThe catalytic domain of SARS-CoV-2 CLpro was crystallized by the hanging drop vapor diffusion method at 296 K with drops consisting of 0.8 µL protein solution (10 mg/mL) and 0.8 µL reservoir solution . These initial crystals were used to make a seed stock using the Glass Seed Bead™ kit . The seeds were prepared from crystals grown in the final crystallization condition. The drop ratios were 0.3 µL protein, 0.2 µL reservoir solution, and 0.1 µL a seed stock dilution of 1/10. Crystals were grown using the hanging drop vapor diffusion method and appeared within 24 h.X-ray diffraction data, collected at a single wavelength on the 7 A beamline at the Pohang Light Source, were processed and scaled using the HKL2000 suite .M  =  2.37 Å3 Da−1). Iterative density modification, model building, and refinement were performed using the AutoBuild wizard of PHENIX. An electron density map was calculated, and an initial model containing four protomers in the unit cell was obtained. Noncrystallographic symmetry (NCS) matrices were obtained for four protomer molecules in the asymmetric unit and were applied during initial refinement. NCS was released earlier to avoid model bias from the NCS average. The model was further refined using COOT [Crystallographic phases for the 2.27 Å catalytic domain of SARS-CoV CLpro dataset were obtained by molecular replacement using the AutoMR wizard in PHENIX . Based oing COOT  and PHENing COOT .work- and Rfree- values of 20.16% and 26.41%, respectively. The stereochemical quality of the final model was evaluated by PROCHECK [The final model of the catalytic domain was refined to RPROCHECK . The RamThe recent pandemic caused by SARS-CoV-2 is slowly settling down. Nevertheless, the occurrence of mutants is still a threat globally. In this study, structural and enzymatic characterization of the catalytic domain of SARS-CoV-2 3CLpro was provided. Intriguingly, the crystal structure of the catalytic domain showed that the major dimeric interface is formed in the unit cell despite the absence of the dimerization domain, domain III. Therefore, the importance of the N-terminal and LH loops was revisited. The N-terminal loop is critical in the formation of a domain-swapped dimer by contributing to the formation of a hydrophobic cavity with the catalytic domain of the other subunit. Surprisingly, the monomeric form of the catalytic domain alone is proven to be enough for its catalytic activity with low efficiency. Sequence and structural analyses showed that the dimerization tendency of 3CLpors is weaker in SARS species. The relatively weak interaction exerted by the DM-loops between domain IIIs seems to be the main reason. Currently, 3CLpro is a good target for the design of anti-SARS-CoV-2 drugs. Some targeting SARS-CoV-2 3CLpro have already been commercialized: paxlovid, molnupiravir, and remdesivir ,12,13. M


Pulses such as beans, chickpeas, peas, and lentils are typically consumed whole, but pulse flours will increase their versatility and drive consumption. Beans are the most produced pulse crop in the United States, although their flour use is limited. To expand commercial applications, knowledge of pulse flour attributes important to the food industry is needed. This research aimed to understand the food industry’s needs and barriers for pulse flour utilization. An online survey invitation was sent via direct email to individuals employed in food companies developing wheat flour products. A survey weblink was distributed by pulse commodity boards to their membership. Survey questions asked food manufacturers about intrinsic factors of pulse flours that were satisfactory or challenging, and extrinsic factors for use such as market demand. Of the 75 complete responses, 21 currently or had previously used pulse flours in products, and 54 were non-users of pulse flours. Ten users indicated that there were challenges with pulse flours while five did not. Two of the most selected challenges of end-product qualities were flavor and texture. Over half of the respondents were unfamiliar with bean flour. Increasing awareness of bean flours and their attributes coupled with market demand for pulse flour-based products may be the most important extrinsic factors to increasing use among food manufacturers rather than supply or cost. Pulses are dry seeds of starchy leguminous crops cultivated worldwide, serving as a source of protein, dietary fiber, vitamins, and minerals ,2. The mNumerous studies have been conducted to investigate the suitability of pulse flours for various food applications with various substitution ratios to wheat flour ,21,22. PThe perspectives of end-users on pulse flours  are scarce in the literature. One case study, conducted among European and multinational food manufacturers of pulse-based products, identified important factors for pulse-based product development . Those fPhaseolus vulgaris, L.) is the most produced pulse crop globally and in the U.S.  mostly confidential for them. A similar observation was made in another pulse products innovation study. Only two firms filed a patent on their new method of pulse processing (which will be published), and all others chose to hold it as a trade secret .Despite pilot testing and expert content review, the survey form may have contained questions or choices that participants did not understand or interpret as intended. Words such as flavor, texture, and dough handling properties could mean various attributes depending on products.An online survey was administered to 75 food industry professionals whose firms produce food products from regular wheat and/or gluten-free flours. The majority of respondents did not use pulse flours. Yeast breads were the most selected product type for which they are using, used, considered using, or would be interested in using pulse flours. Chickpea and pea were the top two pulse flours selected. For the industry personnel who looked for technical information on pulses, scientific associations, both industry and academic, and open-access journals were the main sources. Regarding bean flour in particular, increasing publicity and improving flavor and functionality may be key improvements for inclusion in flour-based food products. Lectin and gluten contaminations were less selected by respondents as concerns. Market demand are likely to be just as important as or more important for continued use of pulse flours than intrinsic characteristics. According to the 21 CPC users, texture, appearance, and uniformity were the three most selected satisfactory characteristics of products using their pulse flour. Ten stated that there were challenges, but five stated there were no challenges. Flavor, texture, dough handling properties, mouthfeel, and volume were the most selected challenges. More than half of the CPC users agreed that pulse flour quality (sometimes) varies from supplier to supplier; however, opinions were mixed about universal specifications for pulse flours and the effect of the variation on the consistency of product quality. Extrinsic factors such as pulse flour supply or cost may not be a main problem to pulse flour users. In summary, flavor, texture, lack of gluten, and market demand were important factors in pulse flour use among food industry professionals who participated in this survey. Individual characteristics that need improvements for specific pulse flour and product type should be studied in future research.