The atomic composition ratio of solution-processed oxide semiconductors is crucial in controlling the electrical performance of thin-film transistors (TFTs) because the crystallinity and defects of the random network structure of oxide semiconductors change critically with respect to the atomic composition ratio. Herein, the relationship between the film properties of nitrate precursor-based indium-zinc-oxide (IZO) semiconductors and electrical performance of solution-processed IZO TFTs with respect to the In molar ratio was investigated. The thickness, morphological characteristics, crystallinity, and depth profile of the IZO semiconductor film were measured to analyze the correlation between the structural properties of IZO film and electrical performances of the IZO TFT. In addition, the stoichiometric and electrical properties of the IZO semiconductor films were analyzed using film density, atomic composition profile, and Hall effect measurements. Based on the structural and stoichiometric results for the IZO semiconductor, the doping effect of the IZO film with respect to the In molar ratio was theoretically explained. The atomic bonding structure by the In doping in solution-processed IZO semiconductor and resulting increase in free carriers are discussed through a simple bonding model and band gap formation energy. Ireported ,6. Partireported ,8,9.−1s−1 and low driving voltages of less than 2 V have been reported [−1s−1 were controlled by varying the ratio of In:Zn or In:Zn:Ga, and the thickness, roughness, and crystallinity of the semiconductor film were investigated relative to the atomic ratio [−1s−1 was demonstrated using various dopant materials [With increasing interest in solution-processed oxide semiconductors, TFTs with high electrical mobilities of over 30 cm·Vreported ,11. Morereported ,13. Amonic ratio ,15,16. Faterials ,18,19,20aterials ,22. In caterials . AlthougC) are caused by weak bonds and structural defects in the fabrication process. As regards the oxide semiconductor manufactured by plasma sputtering, a thin film with a relatively uniform amorphous random network structure can be fabricated on the basis of the composition ratio of source materials, while the crystal structure of the solution-processed oxide semiconductor is difficult to control.Similar to solution-processed oxide semiconductor TFTs, the electrical performance of plasma-deposited ternary or quaternary oxide semiconductor TFTs is significantly affected by the chemical composition of the oxide semiconductor ,25,26. TFor solution-processed oxide semiconductor TFTs, the compositional properties of the solution-processed IZO semiconductor are achieved by eliminating the solvent and precursor, and the electrical properties of the fabricated film are different from those of the plasma-vapor-deposited semiconductors ,15. In tThis study entailed an examination of the doping effect of a solution-processed IZO semiconductor with respect to the In molar ratio by using a simplified atomic bonding structure of the component atoms. Furthermore, the formation energy for each bonding structure was classified into an IZO semiconductor band diagram. For this investigation, a solution-processed IZO TFT based on a nitrate precursor was fabricated with respect to the In molar ratio, and its electrical properties were evaluated. The structural and stoichiometric properties of the IZO semiconductor films were examined based on the electrical properties of the IZO TFTs. Regarding the structural characteristics, IZO films were prepared with respect to the In concentration, and the morphology, thickness, crystallinity, and depth profile of the semiconductor films were assessed using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Auger electron spectroscopy (AES) measurements. Moreover, the stoichiometric properties of the IZO films were determined via thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and Hall effect measurements. Consequently, the performance of the IZO TFT was analyzed in relation to the structural/stoichiometric characteristics of the IZO film, and the electron doping effect with respect to the In concentration was examined based on the atomic bonding structure.3(NO3)3∙xH2O) and zinc nitrate hydrate(Zn2(NO3)2∙xH2O) in 2-methoxyethanol(CH3O(CH2)2OH), 2-ME). For vigorous ionization and hydrolysis reactions, the solution was mixed using a magnetic bar at 60 °C for 12 h or more. In this process, the metal-nitrate precursor cluster forms an azeotrope of nitric acid and H2O through ligand decomposition, and the bonding structure is simplified. The molar ratio and compositional ratio after the deposition of the IZO solution was determined using the ratio shown in The process schematic shown in p-type silicon wafer sputtered with a 100-nm-thick SiNx dielectric layer was cleaned with acetone, isopropyl alcohol, and deionized water using an ultrasonicator for 20 min. After drying, the substrate was treated with oxygen plasma for 1 min at approximately 1 mTorr to lower its surface energy. The IZO film was fabricated on the substrate by covering the prepared solution and spin-coating it for 1 min at 2000 rpm. Soft baking was performed for approximately 5 min on a hot plate at 120 °C, under ambient atmospheric conditions. In this process, nitric acid, an azeotrope of H2O, and the solvent were evaporated, and the IZO solution was gelated in the form of a metal-nitrate-hydrate bond.Before spin-coating of the substrate in −1, and cooling of the substrate was performed for more than 6 h. In the annealing process of the IZO solution, a nitrogen atmosphere was not required but was used to reduce the external interference. The nitrate precursor in the IZO solution was removed in the form of NOx gas through a metal-nitrate thermal decomposition reaction. The thermal decomposition reactions of In and Zn are initiated at 183 °C and 237 °C, respectively, and residual metal-nitrate bonds are removed at a high temperature of approximately 350 °C or higher. After the thermal decomposition reaction, through a consecutive condensation reaction, the hydroxide bond M-OH is discharged into O2 and H2O gas to form an O-M-O oxo linkage structure.Annealing of the IZO semiconductor film was conducted in a nitrogen atmosphere convection oven at 550 °C for 1 h. For annealing, the speed of heating the substrate was controlled at 5 °C min−6 Torr, and the thickness was approximately 50 nm. The IZO TFT was fabricated with a finger-type structure, as shown in On the 20-nm-thick IZO semiconductor film through the annealing process, aluminum source/drain electrodes were thermally deposited through a shadow mask at approximately 1 × 102) using an electron ion beam. The detection elements for the electron ion beam were C, O, N, Zn, In, and Si, and the measuring instrument was an Auger electron spectroscope (AES) (PHI700) . Thermogravimetric analysis (TGA) (TGA N-1000)  to measure the thermal decomposition temperature of the IZO solution was performed in a nitrogen atmosphere using approximately 30 ± 0.5 mg of IZO solution in a platinum pan, and the IZO solution was heated at a rate of 5 °C min−1. For a more detailed analysis of the atomic composition and binding energy of the IZO semiconductor, an x-ray photoelectron spectroscopy (XPS)   was used. In this analysis, XPS measurements of the IZO film were performed after etching the surface by approximately 5 nm with an electron ion beam to prevent surface contamination. For Hall effect measurement, a solution-processed IZO film of TFT structure is fabricated on a substrate cut to 2 × 2 cm2. The carrier concentration, resistivity, and hall mobility of the IZO films with respect to the In molar ratio were analyzed using a Hall effect measurement system (HMS-3000)  by adjusting the input ranges of the current and magnetic field to 1 nA–50 μA and 50 T–0.05 T, respectively.This paper focuses on analyzing the correlation between the electrical properties of IZO TFTs and the fundamental properties of IZO semiconductor films with respect to In concentration. The following equipment and methods were used to measure the electrical properties of the IZO TFT and IZO film. First, the electrical properties of the fabricated IZO TFT were measured using a semiconductor analyzer (ELECS-420)  in an ambient lab atmosphere. The presented electrical data were obtained by coordinating more than 200 measurement results of IZO TFTs. The surface characteristics and thicknesses of the IZO semiconductor thin films were measured using field-emission scanning electron microscopy (FE-SEM) (JS-6701F) . Before the SEM surface image measurement, the surface was coated with platinum (≤5 nm) to prevent electron charging on the surface of the semiconductor film. High-resolution X-ray diffraction (HR-XRD)  was used to analyze the surface and bulk crystal structures of the prepared IZO films. For the analysis, the crystallinity of the surface of the IZO film was measured using the grazing incidence (GI) mode. Furthermore, the depth profile of the IZO film was determined by etching the film from the surface at a speed of 8 nm/min (with reference to SiOG = 40 V and VD = 40 V for IZO TFTs. As shown in D) versus gate voltage and threshold voltage versus In molarity graph. D_on, field-effect mobility μFE) extracted from the transfer curves, where ID_on is the drain current value when VD = 20 V and VG = 40 V of the transfer curve. The electrical parameter values that can be extracted from D)–VG graph in D(sat) is the TFT saturation drain current, μFE is the field-effect mobility, COX is the gate capacitance, and W/L is the channel width and length. Threshold voltage VTH was extracted from the sqrt(ID)–VG graph, as shown in D) also increased with an increasing In molar ratio, and the extracted threshold voltage shifted in the negative direction. As shown in the log-log scale graph in The electrical performance and operating characteristics of solution-processed IZO TFT were evaluated with respect to the In molar ratio. 3N4 lattice characteristic peaks and do not appear in the GI mode because they show surface crystallinity. Several characterized ZnO, IZO, and InO reference crystal peaks are shown at the top of each graph. First, in the crystal properties of the IZO films in The XRD measurement was performed to analyze the crystallinity of the fabricated 20-nm-thick solution-processed IZO film with respect to the In molar ratio. In addition to the structural characteristics of the IZO thin film, its depth profile was measured using AES, as shown in There was no remarkable effect of the In concentration on structural properties, such as roughness, thickness, and crystallinity, and thus, the TGA of the IZO solution was measured to analyze the compositional characteristics of the elements and chemical reaction pathways. The TGA graph measured with respect to In concentration is shown in The electrical characteristics of TFT may also be affected by the distribution of oxygen vacancies in semiconductor films. To analyze the stoichiometric and binding energy characteristics of the IZO film with respect to the In concentration, XPS analysis was performed, as shown in b), Hall mobility (μH), resistivity (s) measured from the Hall effect characteristics. It can be stated that the Hall mobility μH is comparable to that of the field-effect mobility μFE in The Hall effect was measured to investigate the electrical correlation between the electrical characteristics of the IZO TFT and IZO semiconductor film. The results in Summarizing the structural properties  of the IZO film with respect to the In concentration, the IZO film had identical morphology, thickness, and similar crystal properties regardless of the In concentration. In contrast to their similar atomic structural properties, the density and composition ratio were effectively influenced by the In concentration. In addition, the hole mobility and carrier density of the IZO film significantly increased with increasing In concentration. As a result, the change in the electrical characteristics of the IZO TFT with respect to the In concentration in D_on is the saturation current of IZO, μFE is the field-effect mobility extracted from the transfer curve, and Equation (1), where q is the charge amount, ED is the source-drain electric field (VD/L), and A is the cross-sectional area of the drain current . From the estimation of n in Th is expressed as follows:FB is the flat-band voltage, ε0 is the dielectric constant of the vacuum, εs is the dielectric constant of the semiconductor, ΦMS is work function difference, QF is the fixed charge in silicon nitride dielectric, and VF is the Fermi potential; VF = 0.0259 ln (Nb/ni). The IZO semiconductor trap concentration NT was calculated using the relation NT = Qit/qds and the interfacial charge density Qit = qDit. In the calculation of the main parameters Qit and NT, QF/Ci was assumed to be about 30 V; Because the same gate dielectrics were used, it is assumed that the fixed charge value will be similar, and the work function difference is neglected because it is approximately 1 V error. In addition, it was assumed that the charge amount of Qit was uniformly distributed in the ~20 nm of the IZO film. In the analysis of the threshold voltage with respect to the In concentration, the magnitude of Qs/Ci differs by ~24 V for the 9.0 × 1014–8.4 × 1017 cm−3 bulk concentration Nb, and the difference in the VF value is approximately 0.3–0.5 V. The calculated Qit/q values were 5.7 × 1011–5.6 × 1013 cm−2 as shown in T values were 2.8 × 1017–2.8 × 1019 cm−3. Based on the interpretation of Equation (3), the approximately 40 V change in VTh is dominated by a fairly large amount of Qit charge. From the theoretical calculation of VTh, it can be observed that the effect of the trap density is more critical than the variation in the bulk doping for the change in the electrical properties with respect to the In concentration. In this study, NT was estimated by simplifying the Qs and Qit values as much as possible; however, the practical NT values were exponentially distributed from the bandgap EC. Therefore, for accurate threshold-voltage calculations, [T as an exponential function as follows:i is the intrinsic carrier density of the semiconductor, and kT is 0.0259 eV. Assuming that the trap density distribution decreases exponentially from EC, NT0 is the trap density at EC and ET is the characteristic energy that is related to the slope; where the EC was 1.6, which is half of the band gap and VFB in Equation (4) is VFB = ΦMS + QF/Ci by replacing the effect of interfacial charge in NT. According to the calculation of Equation (4), the trap density at EC was distributed as 1019 cm−3 to 1022 cm−3 from EC for an In concentration of 0.0125 M to 0.2 M of IZO TFTs, and the characterization energy was 0.46 to 0.04 eV. A detailed distribution of acceptor-like trap states within the IZO band gap can be estimated through thermal energy analysis and will be introduced through our improved study in our future work. Additionally, in R is the leakage current density between the gate-to-drain electrode and W and ep are the depletion width and emission rate constant, respectively. As the number of traps increases, the number of recombination-generation sites increases, and the leakage current increases.Based on the above results, the electrical characteristics of the carrier concentration in the IZO semiconductor film can be theoretically calculated. The estimated major carrier concentrations for In are summarized in lations, ,39,40 shZnO (DZnO− + VO−) or a donor-like state of oxygen vacancy VO (VO+ + e−) can be created depending on the energy position. Additional reactions, such as charge trapping and detrapping, occurred by VO and DZnO in this model. However, because the electrical conductivity of the ZnO TFT without In was extremely low; it was estimated that the concentration of this bonding structure was relatively low. Furthermore, the In-doping model shown in InO− are generated to sustain the net equilibrium state. A considerable amount of the acceptor-like trap DInO− is ionized and electrons (DInO−→DInO0 + e−) become free electrons, forming a neutral state as a whole. As the In concentration increased, the Zn atoms in the ZnO structure were replaced by In atoms, thereby increasing the IZO film density and increasing the total concentration of free electrons.The following results were inferred based on the structural/stoichiometric properties of the IZO semiconductor and the electrical properties of the IZO TFT. The role of ZnO in the solution-processed IZO semiconductor film is presumed to form an amorphous random network structure during the fabrication process. In addition, it can be determined that the role of In atoms in the solution process is to increase the free carrier density of the IZO semiconductor by structurally replacing Zn atoms. From the analysis results of the threshold voltage, it was speculated that these free carriers were primarily influenced by the trap charge. Based on the observed results, a simplified atomic model for In doping is shown in ZnO, DInO) can be positioned inside the band gap. Finally, C) and the valence band maximum (EV) of the IZO semiconductor were defined by considering the In 5s and O 2p orbitals, and these energy positions were sketched based on [C of the band gap, as shown in C, and the Zn-O defects will be relatively deep and have low concentrations. In addition, note that the distribution of acceptor-like states inside the bandgap is determined not only by the defect traps but also by the weak bond between the Zn-O and In-O structures in Zn0 of Zn-O is replaced by the In-O formation energy DIn−, the subsequent density of the shallow state increases, and the density of the deep state decreases. Therefore, the EF level of the IZO semiconductor shifts towards the conduction band EC [Considering the above results, based on . The for band EC ,46. AlthIn this study, we suggested the simplified atomic bonding structure and In doping model in the energy band diagram for solution-processed IZO semiconductors. For this investigation, the structural characteristics of the IZO semiconductor and the electrical characteristics of the IZO TFT were analyzed with respect to the In molar ratio. The optimal characteristics of the IZO TFT were obtained at the composition condition of the In:Zn ratio of 14.4:16:3, which were found to be comparable to the results reported in the literature. Moreover, the electrical conductivity of the IZO TFT increased significantly with increasing In concentration, and the threshold voltage shifted in the negative direction. The structural characteristics of the IZO film were analyzed by SEM, XRD, and AES, and no specific trends with respect to the In concentration were observed in the IZO films. The fabricated IZO thin film had a multilayer structure; the bottom layer was IZO, and the surface was formed of a ZnO aggregation layer. Although the IZO films did not show significant structural changes with respect to In concentration, the stoichiometric properties of the IZO films changed significantly. These changes were demonstrated through TGA, XPS, and Hall effect measurements. The structural density and In composition ratio of the films significantly increased with respect to the In molar ratio, and the subsequent carrier concentration of the bulk film remarkably increased. Herein, despite the structural similarity of the IZO film, the enhancement in the electrical properties of the IZO TFT can be explained by the increase in the carrier concentration with respect to the In concentration. Consequently, it is reasonable to conclude that the donor doping effect by these carriers contributes to an increase in the acceptor-like states owing to structural defects, which was proposed as a model for the atomic bonding structure and trap state of IZO semiconductors in this study. We believe that analyses of the bonding structure between atoms and the energy structure thereof are essential to understand the change in electrical properties according to the material composition of the solution-processed oxide semiconductor. This study can be used in future studies to improve the performance of solution-processed oxide TFTs by combining different material compositions and doping techniques.


Reversion of the wild-type protein sequences of single transmembrane melanocortin accessory protein families (MRAP2) in mice and zebrafish created novel functional pharmacological modulators for regulating melanocortin 4 receptor (MC4R) signaling. All of the brand new reversed MRAP2 (rMRAP2) proteins could form proper dimeric topology on the plasma membrane and interact with and affect the ligand-stimulated pharmacological profiles of zebrafish and mouse MC4R signaling in vitro.As a member of the melanocortin receptor family, melanocortin 4 receptor (MC4R) plays a critical role in regulating energy homeostasis and feeding behavior, and has been proven as a promising therapeutic target for treating severe obesity syndrome. Numerous studies have demonstrated that central MC4R signaling is significantly affected by melanocortin receptor accessory protein 2 (MRAP2) in humans, mice and zebrafish. MRAP2 proteins exist as parallel or antiparallel dimers on the plasma membrane, but the structural insight of dual orientations with the pharmacological profiles has not yet been fully studied. Investigation and optimization of the conformational topology of MRAP2 are critical for the development of transmembrane allosteric modulators to treat MC4R-associated disorders. In this study, we synthesized a brand new single transmembrane protein by reversing wild-type mouse and zebrafish MRAP2 sequences and examined their dimerization, interaction and pharmacological activities on mouse and zebrafish MC4R signaling. We showed that the reversed zebrafish MRAPa exhibited an opposite function on modulating zMC4R signaling and the reversed mouse MRAP2 lost the capability for regulating MC4R trafficking but exhibited a novel function for cAMP cascades, despite proper expression and folding. Taken together, our results provided new biochemical insights on the oligomeric states and membrane orientations of MRAP2 proteins, as well as its pharmacological assistance for modulating MC4R signaling. Activation of MCRs leads to adenylate cyclase stimulation and accumulation of the intracellular cyclic adenosine monophosphate (cAMP), with downstream signaling cascades. These five members distribute throughout the body and play distinct roles in regulating physiological processes by selectively binding to various melanocortin peptides ,3,4,5. A(ERK1/2) . Intrigu(ERK1/2) .MRAP2-deficient mice develop severe obesity, which is presumably resulting from a significant decrease in MC4R sensitivity to α-MSH in the absence of MRAP2 protein [Xenopus laevis and tropicalis (African clawed frog), MRAP and MRAP2 both exert potentiation patterns on modulating MC3R and MC4R signaling, stimulated by α-MSH and ACTH [Danio rerio (zebrafish), Mrap2 exists in two isoforms, Mrap2a and Mrap2b. They express at different time points over the course of embryonic development and exhibit distinct functions on modulating Mc4r signaling. Simply, Mrap2a mainly expresses in larvae and facilitates zebrafish growth by specifically blocking the action of Mc4r, while Mrap2b expresses in a later developmental stage and increases the pharmacological sensitivity of Mc4r to the endogenous ligand [Melanocortin 2 receptor accessory protein (MRAP) functions as a small protein with a single membrane-spanning domain and conserved N- and C-terminals. It was originally identified as an MC2R chaperone with indispensable roles for the trafficking, ligand binding as well as the signal transduction of MC2R . As a pa protein . In the and ACTH ,29,30. Is ligand ,32.Previous studies found that mammalian MRAP2 existed on cell membranes as parallel or antiparallel homodimers, and it could also form higher-order oligomers ,33,34. Shttps://www.ebi.ac.uk/Tools/msa/muscle/ (accessed on 26 June 2021). The alignment results were presented with MView. Available online: https://www.ebi.ac.uk/Tools/msa/mview/ (accessed on 26 June 2021), where amino acids were labeled by different colors, according to their conservative property. TM prediction was accomplished by TMHMM Server v. 2.0. Available online: http://www.cbs.dtu.dk/services/TMHMM/ (accessed on 18 December 2021).The multi-sequence alignment shown in mc4r and mrap2a/b, and mouse Mc4r and Mrap2 were cloned from zebrafish and mouse brain cDNA, respectively. Reversed mrap2a/b (Rmrap2a/b) and reversed Mrap2 (RMrap2) were synthesized from GENEWIZ company . All the genes above were inserted in the pcDNA3.1(+/−) vector, with 3xHA or 2xflag tag added at the 5′-end. For the bimolecular fluorescent complimentary (BiFC) assay, the YFP-F1/F2 sequence was, respectively, fused to the 3′-end or 5′-end of Rmrap2a/b and RMrap2.Zebrafish 2. Transfection was carried out with polyethylenimine L (PEI)  until cells grew to 70–80% confluency. The indicated plasmids were mixed in serum-free DMEM and PEI solution was added to the plasmid mixture at a 4:1 ratio, and then it was gently pipetted to mix the solution. Finally, the mixture was applied into cells for 15 min incubation.Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) , containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, at 37 °C with 5% COMc4r:RMrap2 = 1:1 or Mrap2:Rmrap2 = 1:1, and the DNA total amount was kept at 2 µg for each well in 6-well plates), DMEM was removed and 400 µL of IP cell lysis buffer  was added to each well. Next, the cell lysis was transferred to 1.5 mL tubes and rotated for 1 h at 4 °C. The cells were then centrifuged for 15 min at 4 °C and 17,000× g. The supernatant was transferred to a new tube. A total of 40 µL of the supernatant was retained as a control and the rest of the specimen was incubated with anti-HA-Mouse  overnight at 4 °C. Total protein was stored at −20 °C upon the addition of protein loading buffer. The next day, Protein A+G Agarose  beads were added to the IP tubes and rotated for 4 h at 4 °C. The beads were then washed three times with cell lysis buffer containing protease inhibitor and centrifuged at 4 °C and 1000× g to discard the supernatant. Finally, 40 µL of 1× protein loading buffer was added to resuspend the beads. Both the total and the IP protein were boiled at 95 °C for 15 min. For SDS-PAGE, 12% gel was utilized and 10 µL of each sample was loaded. HA-Rabbit (1:5000)  and DYKDDDDK (Flag)-Rabbit (1:5000)  antibodies were utilized to blot MC4R and RMRAP2 or other proteins, respectively.Between 24–48 h after transfection . Between 24–48 h later, the media was removed, and 4% paraformaldehyde was added to fix the cells. Anti-HA and anti-Flag antibodies (1:1000) were applied to incubate the cells for 2 h at room temperature, and then, the cells were washed three times with DPBS . Alexa Fluor488 and 647 IgG  were employed as secondary antibodies for HA and Flag, respectively. After incubation with fluorescent secondary antibodies for 2 h, protected from light, the cells were washed three times with PBST. Next, ProLong(R) Gold Antifade with DAPI Molecular Probes  was applied on the glass slides and covered by transparent slides. Finally, the slides were observed with a Carl Zeiss confocal laser scanning microscope under a 63x oil lens.Cells were seeded in Poly-D-lysine pre-treated 12-well plates with slides and transfected with the indicated plasmids  the following day. Between 24–48 h later, the media was removed and 4% paraformaldehyde was added to fix the cells. The cells were then washed three times with DPBS and ProLong(R) Gold Antifade with DAPI Molecular Probes was applied on glass slides and covered by transparent slides. Finally, the slides were observed with a Carl Zeiss confocal laser scanning microscope under a 63× oil lens.-mMc4r and 2xFlag-RMrap2 (or homologous zebrafish genes) at ratio 1:0, 1:3 and 1:6 . The amount of Mc4r was fixed and the rest was complemented with blank pcDNA3.1 vector. Between 24–48 h later, the DMEM was removed and 4% paraformaldehyde was added to fix the cells. After blocking with 5% milk in PBS, the cells were incubated with anti-HA antibody (1:2000) for 2.5 h at room temperature and washed three times with DPBS. Anti-IgG secondary antibody  was added to each well and incubated for 2 h at room temperature, and then the cells were washed three times with DPBS. Next, the cells were incubated with tetramethylbenzidine (TMB) substrate  for 15–30 min at room temperature, protected from light. Finally, the supernatant was transferred to a clear 96-well plate containing 2 M sulfuric acid and absorbance was measured with an ELISA reader (SpectraMax iD3) at OD = 450 nm. Janus Green was utilized to normalize the cell numbers.The cells were seeded in Poly-D-lysine pre-treated 24-well plates and transfected with 3xHAmMc4r and RMrap2 (or zebrafish homologous genes). The DNA total amount was kept at 0.25 µg for each well in the 24-well plates. The amount of Mc4r was fixed and the rest was complemented with blank pcDNA3.1 vector. Between 24–48 h later, the medium was replaced by fresh DMEM containing 0.1% bovine serum albumin (BSA) as well asα-MSH, ranging from 10−12 M to 10−6 M. After a 4 h incubation, the Dual-Glo Luciferase Assay System  was applied to measure the cAMP level, according to the manufacturer’s instructions. The cell mixture was transferred to white 96-well plates and the firefly luminescent intensity was measured on the ELISA reader (SpectraMax iD3) with the Dual-Glo luciferase protocol. The Renilla was measured to normalize the cell numbers upon the addition of stop solution. For antagonism experiments, the cells were stimulated by 1 × 10−8 M α-MSH for zebrafish and 1 × 10−7 M α-MSH for mice, with a concentration of SHU9119 ranging from 10−12 M to 10−6 M. Three biological replicates (independent transfections) were performed for each group.The cells were seeded in 24-well plates and transfected with a mixture of Renilla , Pcre-luc (to measure cAMP induced by Gs), p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.All the experiments were conducted at least three times. Data were analyzed using GraphPad Prism 8 . cAMP assay results were analyzed by nonlinear regression (curve fit). The results of the surface ELISA were analyzed by a one-way ANOVA. All the results were shown as mean ± SEM. ns (not significant), * mrap2a/b and 3xHA-mc4r were transfected into HEK293T cells and co-immunoprecipitation (CoIP) assays found that RMrap2a/b interacted with zMc4r  and zebrafish Mrap2a/b (zMrap2a/b) and artificially constructed reversed MRAP2 protein sequences . Multi-sth zMc4r D,E and tth zMc4r F. Receptth zMc4r C,G becauth zMc4r C–F. Nextth zMc4r F–H, furtIn our previous study, we found that Mrap2a and Mrap2b could not only form parallel and antiparallel homodimers on plasma membranes but also form parallel and antiparallel heterodimers with each other . To testTheoretically, RMRAP2s should exhibit the same dual topology as WT ones because of the retention of a transmembrane domain, meaning that the dimers could be formed both parallelly and antiparallelly A. To expSubsequently, the pharmacological function of RMRAP2s was further assessed by examining their assistance to zMc4r and mMC4R signaling. Our previous study showed that Mrap2a and Mrap2b distinctly influenced the surface expression of zMc4r in the zebrafish , where zmc4r and mrap2a or mrap2b were transfected into cells at different ratios . Then, these cells were stimulated by α-MSH or suppressed by SHU9119 at different concentrations (10−12–10−6 M). Different from WT zMrap2a, which inhibited zMc4r action [The pharmacological activity and intracellular signal transduction of MC4R is significantly influenced by MRAP2. Therefore, it is essential to assess the function of RMRAP2s in this aspect. The common ligands of MC4R include ACTH (agonist), α-MSH (agonist), Agouti-related protein (AgRP) (antagonist) and SHU9119 (antagonist) . The actr action , zRMrap2r action , the senp < 0.05, ** p < 0.01, *** p < 0.001.Data shown in Data shown in mc4r and mouse Mc4r were transfected into HEK293T cells along with different ratios of reversed mrap2a/b and Mrap2 . As indicated in The reversion of MRAP2s produces a novel modulator with new functions. It is interesting to investigate how MC4R is affected when WT and reversed MRAP2s coexist. Zebrafish As a key member of the MCR family, MC4R plays crucial roles in regulating energy homeostasis and feeding behavior. Therefore, MC4R has become a promising GPCR drug target for severe obesity therapy. Setmelanotide, synthesized as an agonist for MC4R, was approved by the U.S. Food and Drug Administration (FDA) in the United States in November 2020, and in the European Union in July 2021, for alleviating severe obesity syndrome. Because of the significant sensitization of MC4R cascades by MRAP2 proteins, it is plausible to enhance the agonist stimulation through conformational allosteric modulation. Therefore, it is meaningful and valuable to explore the regulatory mechanism of MRAP2 for the modulation of MC4R signaling. Functional MRAP2 complex exists on the plasma membrane as dimers or oligomers. Given the presence of antiparallel homodimers of MRAP2, we want to uncover the difference between two orientations of MRAP2, either the N-terminus orientating intracellularly or extracellularly. To test this hypothesis, zebrafish RMrap2a, zebrafish RMrap2b and mouse RMRAP2 were synthesized and the dimerization, protein interaction and pharmacological activities on MC4R signaling were assessed in this study.It has been reported that reversed protein sequences are perhaps more foldable than native ones and the ability of reversed sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core . MRAP2 fDespite their native-like structure, mouse and zebrafish RMRAP2s differ from WTs in the regulation of MC4R trafficking and signaling. RMrap2a shows elevating regulation of Mc4r signaling, which is opposite to the effect of Mrap2a . Unlike Combining the results of surface translocation and pharmacological modulation, it can be proven that different parts of MRAP2 own different functions, and the domain for assisting MC4R trafficking and the one influencing MC4R signaling are in distinct locations. This finding is similar to the analysis concluded by Rouault et al.  in whichWhen MRAP2s and RMRAP2s were co-expressed in the same cells, we found that the sensitizing effect of RMrap2a dominated despite the presence of the blocking effect of Mrap2a. In contrast, Mrap2b and RMrap2b showed equal assistance for MC4R signaling. The opposite modulation of Mrap2a’s N-terminus and C-terminus in MC4R signaling was a hint for identifying precise motif enhancing or blocking Mc4r signal transduction.In conclusion, our results confirmed the symmetry of the MRAP2 transmembrane domain and revealed the non-random orientation of MRAP2 proteins. Surface expression and CRC assay identified reversed Mrap2a and Mrap2b as new functional modulators, exerting a novel impact on regulating Mc4r trafficking and signaling. These findings will help us to further understand the asymmetry of the opposite ends of MRAP2 and its dual orientation on the plasma membrane, and may provide scientific inspiration for how to create a novel accessory and allosteric modulator for melanocortin receptors or other GPCRs in the future.


Scanning microwave microscopy (SMM) is a novel metrological tool that advances the quantitative, nanometric, high-frequency, electrical characterization of a broad range of materials of technological importance. In this work, we report an inverted near-field scanning microwave microscopy (iSMM) investigation of a graphene oxide-based epoxy nanocomposite material at a nanoscopic level. The high-resolution spatial mapping of local conductance provides a quantitative analysis of the sample’s electrical properties. In particular, the electrical conductivity in the order of ∼ The scanning microwave microscope (SMM)  is one oImtiaz and Anlage  combinedctometry . This mectometry .More recently, our group developed a new setup called the inverted SMM (iSMM) , which imeasured ,15,16. Imeasured . Furthermeasured , which ameasured  or subcemeasured  in a phymeasured .The electrical and electronic applications of graphene-based composites have been gaining attention for their wider range of applications in industries . These cThe iSMM setup is basically composed of an AFM (NT-MDT Solver Pro-47) and a planar transmission line, which also serves as a sample holder. The slot line is deposited onto a 30 mm × 30 mm low-loss laminate substrate with a thickness of 512 µm. The metallic part is composed of a layer of copper (30 µm) and nickel (5 µm), with a 30 nm gold coating on top. The top part of the line is made to have a smooth surface with a very low roughness, which is a requirement for the scanning of the nanoparticles with AFM. The signal line has a width of 200 µm, with a gap of 100 µm to the ground line. The slot line is designed to have resonances by introducing a transition, from the typical system impedance of A 3D model of the slot line is simulated with a commercial finite-difference time-domain (FDTD)-based electromagnetic solver CST Microwave studio. The resulting electric field map at 6 GHz is shown in The sensitivity of the system for the optimal working frequency range for an iSMM scan can first be identified by predicting the signal-to-noise ratio (SNR), as defined in Equation  expresseTypically, good quality iSMM images can be obtained with an SNR over 20 dB. The sample material is a two-component epoxy system. The epoxy resin was supplied by Sicomin and is composed of an SR 1660-type resin and an SD 2630-type catalyst; it is characterized by good chemical resistance, oxidation resistance, and a low exothermic peak reinforced with a hardener dispersed into the matrix. This type of resin is commonly used in the manufacturing of parts or tools that operate under high-temperature environments of up to 160 °C. Graphene Oxide (GO) in the form of flakes was supplied by Hygraner.An epoxy-based carbon composite system containing 3.0 wt % of GO was prepared by adding GO to the epoxy resin and dispersing under mechanical stirring. The concentrations of the GO were calculated from the total weight of the reactive system (epoxy resin + hardener). The nano-filled epoxy samples underwent an oven-curing process by applying the following thermal cycle : 48 h atThe fractured surfaces of the manufactured Epoxy/GO were analysed by FESEM  after gold sputtering and with an accelerating voltage of 5 kV. The fractured surfaces of the Epoxy and the Epoxy/3%GO were gold-coated with an Agar automatic sputter coater and then analysed.The compactness of the epoxy systems was investigated by observing the internal surfaces. The inter-facial status of the epoxy and the fillers play a key role in the overall performance of the nanocomposite systems . The effThe iSMM scan was performed in the frequency band of 5.9 to 6.4 GHz, and it recorded the reflected microwave signal / formula :(2)ϵcontHere, terature  was modiThe obtained dielectric constant map of the non-contact case using Equation  is shownIn this work, an investigation of the iSMM system for the quantitative mapping of local electrical properties of a graphene oxide/epoxy nanocomposite was conducted by measuring the local admittance at a high spatial resolution. The electrical conductivity and dielectric constant of the composite material were found to be at ∼


The protein arginine methyltransferase 5 (PRMT5) enzyme is responsible for arginine methylation on various proteins, including histone H4. PRMT5 is a promising drug target, playing a role in the pathomechanism of several diseases, especially in the progression of certain types of cancer. It was recently proved that the phosphorylation of PRMT5 on T80 residue increases its methyltransferase activity; furthermore, elevated levels of the enzyme were measured in the case of human hepatocellular carcinoma and other types of tumours. In this study, we constructed the complexes of the unmodified human PRMT5-methylosome protein 50 (MEP50) structure and its T80-phosphorylated variant in complex with the full-length histone H4 peptide. The full-length histone H4 was built in situ into the human PRMT5-MEP50 enzyme using experimental H4 fragments. Extensive molecular dynamic simulations and structure and energy analyses were performed for the complexed and apo protein partners, as well. Our results provided an atomic level explanation for two important experimental findings: (1) the increased methyltransferase activity of the phosphorylated PRMT5 when compared to the unmodified type; (2) the PRMT5 methylates only the free form of histone H4 not bound in the nucleosome. The atomic level complex structure H4-PRMT5-MEP50 will help the design of new inhibitors and in uncovering further structure–function relationships of PRMT enzymes. Post-translational modification (PTM) is a fundamental mechanism occurring on proteins of different roles in epigenetic regulation ,2,3,4. HProtein arginine methyltransferases (PRMT) are writers that add methyl groups to the N-terminal arginine residues of H4 (or other substrates) . It was PRMT5 is a member of the PRMT family, catalysing the arginine monomethylation and monomethylation of several non-histone and histone proteins, including histone H2A ,11,12, HThe phosphorylation and dephosphorylation of tyrosine ,18 and tThe experimental atomic resolution structure of human PRMT5 in complex with MEP50, a methyl-donor ligand and an eight-amino-acid-long histone H4 fragment, was revealed 10 years ago . Some yeP), as well as for the inactivity of PRMT5 on nucleosome-bound histone H4.The aim of this study was to construct the human PRMT5-MEP50 structure in complex with the full-length histone H4 peptide in order to provide a structural explanation for the increased methyltransferase activity of the T80-phosphorylated enzyme (PRMT5The explanation of the difference in enzymatic activity (activation energy) of the two PRMT5 variants necessitates the atomic resolution structures of the H4-PRMT5 complexes. However, PRMT5(-MEP50) in complex with the full-length histone H4 has not been measured yet see A is avaiP), it can be expected (Introduction) that the activation energy barrier will decrease  between H4 and PRMT5(-MEP50) was calculated for all snapshots of the MD simulation  from both P1 and P2 plateaus and analysed. The interactions between the catalytic domain of PRMT5 and histone H4 N-terminal residues remained stable throughout the MD simulation. At the same time, a considerable change of the H4 structure was observed between the activated (P1) and pre-product (P2) states, also reflected by the aforementioned drop in the total Einter B. In theinter in the pre-product H4-PRMT5 complex structure  was also performed to estimate the time scale of conformational changes of H4 binding to PRMT5. Considerable changes of the bound structures were measured in terms of Cα RMSD values  of 7.7  that is P necessitates the building of the atomic resolution structure of the H4-PRMT5P-MEP50 complex for a comparison with the unmodified PRMT5 version that is described in P-MEP50 complex was constructed by adding a phosphate group to residue T80 of the energy-minimized H4-PRMT5 complex structure (Methods). The complex was energy-minimized and subjected to a 720 ns-long MD simulation, and Einter was calculated between H4 and PRMT5P-MEP50 along the whole trajectory  residue of PRMT5 and histone H4, as reflected by the per-residue Einter analysis , was used as the starting structure (4gqb). The missing amino acids of PRMT5-MEP50 were built by SWISSMODEL online server , and teri is the atomic mass, and ri(t) is the position of atom i at time t.The root mean square deviation (RMSD) for Cα atoms was calculated during the MD simulation to check the equilibration of the structures. The RMSD of MEP50 fluctuated between 1.5 and 2.0 A , showinggmx rms) and RMSF (command: gmx rmsf) calculations. The RMSF was calculated by Equation (2):i is the position of the particle i, T is the time of the MD simulation, and ref denotes the reference position of the particle i.The root mean square fluctuation (RMSF) was also calculated for each residue in the case of the apo PRMT5-MEP50 and unbound H4 structures, after the snapshots were fitted to the same structures like in the RMSD calculation. GROMACS  was usedP complex, the phosphate group was covalently attached to T80:PRMT5 by PyMol [To build the phosphorylated H4-PRMT5by PyMol . The parby PyMol .−1 nm−2, respectively.Complexes and peptides were submitted to a two-step (steepest descent and conjugate gradient) energy minimization procedure before the MD simulation by GROMACS . Molecul−5 bar−1, and reference pressure of 1 bar. Particle mesh Ewald summation was used for long-range electrostatics. Van der Waals and Coulomb interactions had a cut-off at 11 Å. Periodic boundary conditions were treated after the finish of the calculations. After each trajectory, the periodic boundary effects were handled, the system was centred in the box, and the target molecules in subsequent frames were fit on the top of the first frame. The final trajectory, including all atomic coordinates of all frames, were converted to portable xdr-based xtc binary files.The complex, the unbound histone H4, and the apo PRMT5-MEP50 were submitted separately to a 720 ns-, a 1000 ns-, and a 580 ns-long MD simulation, respectively. In all cases, a TIP3P  explicitinter).ij is the potential well depth at equilibrium between the ith (substrate) and jth (enzyme) atoms; ε0 is the permittivity of vacuum; εr = 1, relative permittivity; Rij is the inter-nuclear distance at equilibrium between ith (substrate) and jth (enzyme) atoms; q is the partial charge of an atom; rij is the actual distance between the ith (substrate) and jth (enzyme) atoms; NE is the number of enzyme atoms; NS is the number of substrate atoms.0 − A, ε0 is the dielectric constant of water at 25 °C, and A, λ, and k are constants.The sum of Lennard-Jones (LJ) and Coulomb (Cb) intermolecular interaction energies were calculated  (3). Theinter values at both the enzyme and the substrate sites for the unmodified and phosphorylated H4-PRMT5 complexes were chosen and merged to prepare the Einter bar chart (The top ten residues with the lowest Ear chart .Representative structures were selected using a structure-based clustering from the following four sets of structures: (i) unmodified H4-PRMT5 complex structures from the P1 and (ii) P2 plateaus; (iii) phosphorylated H4-PRMT5 complex structures from the P1 and (iv) P2 plateaus. The clustering procedure contained the following steps: The average atomic coordinates were calculated for all four set of structures using a bash script, which prints the x, y, and z atomic coordinates of all structures in the set into separate text files. The atomic coordinates were structured into a pdb file format and used as average structures. Finally, the RMSD values between the average structure and each complex were calculated by an in-house program, rmsd, and the structure with the lowest RMSD value was selected as the representative structure.inter-based selection of representative structures was also performed in the pre-product state (P2), as Einter should have a similar value in the unmodified and phosphorylated structures. Accordingly, representatives for the P2 section were determined by calculating the Einter-differences of the unmodified H4-PRMT5 and H4-PRMT5P complexes of the last twenty-five frames. Structures with the lowest Einter-difference were chosen as unmodified and phosphorylated H4-PRMT5 representatives for the P2 section  containA three-dimensional structure of the unmodified and phosphorylated human PRMT5-MEP50, in complex with the full-length histone H4 protein, was modeled. Molecular dynamic simulations and subsequent analyses provided an atomic level explanation for two important experimental findings: (1) the increased methyltransferase activity of the phosphorylated PRMT5 when compared to the unmodified type ; (2) the