Due to the coordination of phosphate groups to Zn(II), N-phosphopeptides can be effectively captured under neutral conditions. Moreover, the method is successfully applied to an E.coli and HeLa N-phosphoproteome study. These results further broaden the range of methods for the discovery of N-phosphoproteins with significant biological functions.Protein N-phosphorylation plays a critical role in central metabolism and two/multicomponent signaling of prokaryotes. However, the current enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides due to the intrinsic lability of P-N bond under acidic conditions. Therefore, the effective N-phosphoproteome analysis remains challenging. Herein, bis(zinc(II)-dipicolylamine)-functionalized sub-2 μm core-shell silica microspheres -dipicolylamine) in N-phosphopeptides effective enrichment. Besides the most recorded O-phosphorylation on serine, threonine, and tyrosine, the phosphorylation of 1-histidine (1-pHis), 3-histidine (3-pHis)4, lysine (pLys)6, and arginine (pArg)8, termed as N-phosphorylation  phosphorylates arginine residues of the heatshock regulator (CtsR) in the DNA binding domain, to regulate gene transcription13. The first documented pLys was found in bovine liver, dating back to 1960s16. However, still little is known about its prevalence and biological roles18.Protein phosphorylation plays important roles in the regulation of signal transduction and gene expression20.The main reason that hinders the study of N-pho is derived from the intrinsic lability of P–N bond. Since the phosphorus d-orbital of π-bonding is in a higher energy shell, the overlap between nitrogen lone pair orbital and the phosphoryl π-bond is scarce. Therefore, the P–N bond is not benefited from the stabilization caused by electronic delocalization. In addition, the basic of nitrogen is preserved, which markedly enhances the leaving ability of the amino group once protonated21, and LHPP is discovered as the protein histidine phosphatase and tumor suppressor22. Likewise, McsB/YwIE is the well-known arginine kinase/phosphatase pair23. However, neither pLys kinases nor phosphatases were found so far, and histone H1 was the only reported pLys protein24.In modern biological approaches, studies were mainly focused on finding target proteins such as kinases and phosphatases. NME1 and NME2 are the only two mammalian protein histidine kinases reported so far26. Fuhs et al.27 generated the first pHis monoclonal antibody, and some pHis candidates related to cell cycle functions were discovered in mammalian cells. Fuhrmann et al.28 developed two isosteres of pArg and generated the first high-affinity pan-pArg antibody for screening the chaperone proteins, ClpC and GroEL in B. subtilis DywlE strain. Hunter group identified 425 N-pho sites from HeLa lysates using hydroxyapatite and 1, 3-pHis monoclonal antibodies (HAP/pHis mAbs)29. However, the structural multiformity introduced by adding phosphate groups to diverse residues makes it rather difficult to develop highly efficient pan-specific antibodies for the global study of N-phosphoproteome. Besides, specific N-pho variants are usually predisposed by certain antibodies and thus can cause biases. For N-phosphoproteome analysis, antibody-independent methods can solve the above problems. However, due to the instability of P–N bond under acidic conditions, the currently used enrichment methods for O-phosphopeptides are not preferred for N-phosphopeptides, resulting that the landscape of N-phosphoproteome was still covered31. Inspired by naturally occurring phosphatases, which provide specific Zn(II)-central enzymatic pockets to bind phosphate units of substrates, bis(zinc(II)-dipicolylamine) molecular (DpaZn) is designed for phosphate targets recognition under neutral conditions32. Moreover, phos-Tag beads with Zn(II) were used for enrichment of non-canonical phosphorylation peptides34, which further demonstrated the reliability of 2Zn(II). Therefore, DpaZn molecular functionalized materials show great potential for N-phosphopeptides enrichment under neutral conditions.To globally elucidate one kind of N-pho, much effort has been made in preparing effective antibodies. Nevertheless, the acid instability and structural flexibility of N-pho makes it difficult to induce immunization by classical methods. Recently, benefited from stable pHis and pArg analogs, several antibodies have been prepared2@DpaZn) are designed for on-tip N-phosphopeptides enrichment under neutral conditions. A total of 27 N-pho sites are identified from Escherichia coli. In addition, SiO2@DpaZn are applied for the analysis of mammalian N-phosphopeptides. In total, 3384 N-pho sites, containing 611 pHis, 1618 pLys, and 1155 pArg, are identified from HeLa cell lysates. We provide reliable technical support for N-pho studies.In this work, DpaZn-functionalized sub-2-μm core–shell silica microspheres (SiO32. Each Zn(II) ion is coordinated with two pyridyl nitrogen atoms, a tertiary amine, a phenoxy anion, and an oxygen atom from phosphate group -chelated bis (dipicolylamine) (Dpa) groups, terms DpaZn, could bind with phosphorylated target (including both N-phosphorylated and O-phosphorylated targets) and form the stable 1:1 complexoup Fig.  inset. H35 Therefore, rapid enrichment is preferred to improve the recovery of N-phosphopeptides36. Herein, sub-2-μm core–shell SiO2 microspheres were designed as the substrate, not only to achieve the specific enrichment within a shorter time, contributed by the fast mass transfer, but also to provide adequate reaction sites for DpaZn immobilization, ensured by the large specific surface area. Accordingly, we could expect that the combination of DpaZn functional groups and core–shell silica microspheres might be effective for the capture of N-phosphoproteome.Moreover, it is noticed that the P–N bond is also heat-labile even under neutral conditions2@DpaZn were prepared by seed growth and template growth methods according to our previous work37, followed by carboxyl terminal Dpa immobilization and two Zn(II) ions coordination  under neutral condition  were strictly checked according to the rules shown in Supplementary Fig. 39. To further explore the influence of different carbon sources on N-pho sites in E. coli, N-phosphopeptides from M9 minimal medium-cultured E. coli (glucose or glycerol) were enriched by our method. After removing the pLys/pArg peptides localized to the C-terminus, 99 N-pho sites  and 101 O-pho sites  with localization probability over 0.75 were identified  unstable to acid41. However, the exact modified sites were still unknown. In this study, two N-pho sites (H26 and K48) on YfiD were identified (AEAGIVISASpHNPFYDNGIK and AGYAEDEVVAVSpKLGDIEYR). The MS/MS spectra of the peptides from in vivo (AEAGIVISAS+79.98DaHNPFYDNGIK and AGYAEDEVVAVS+79.98 DaKLGDIEYR) with a mass shift of +79.98 Da at the histidine and lysine residues had the same MS/MS spectra as that of the synthetic peptides with a phosphate group on histidine and lysine, respectively (AEAGIVISASpHNPFYDNGIK and AGYAEDEVVAVSpKLGDIEYR)  Fig. . In addiYR) Fig. , confirm2@DpaZn was applied for HeLa cell lysates. Totally, 3384 N-pho sites, containing 611 pHis, 1618 pLys, and 1155 pArg, and 6635 O-pho sites, containing 4105 pSer, 1956 pThr, and 574 pTyr, were identified from HeLa lysates  and phosphate groups. As shown in Fig. 3·H2O, non-phosphopeptides, mono-phosphopeptides  and the multi-phosphopeptide (m/z 3122) were eluted in sequence, in accordance with our expectation. Moreover, we found the zeta potential of SiO2@DpaZn kept stable at about +40 mV around pH 7.0  experiments. The interaction between DpaZn and N-phosphopeptides was endothermic, indicating it an entropy-driven process  ion that the phosphate anion [RO(N)PO3] can access to form RO(N)PO3-DpaZn complex. Compared with P–O bond, the N atom of P–N bond has a stronger electron donating ability than the O atom, which makes the structure of RNPO3-DpaZn more stable. Therefore, this might result in N-pho peptides being preferentially enriched over O-pho peptides under the neutral conditions. This also explained the relatively high proportion of N-pho peptides in our results.In this study, SiO3+-IMAC-based strategy for the analysis of pHis proteome in E. coli cultured with glucose and glycerol45. Combining mild acidic (pH 2.3) enrichment and pollutant removal strategy including protein precipitation and benzonase treatment, 135 pHis sites were identified , 6  and 1 (R1879) pArg sites were exclusively identified by SAX, SiO2@DpaZn, and HAP/pHis mAbs, respectively.In additional, a strong anion exchange-based method (SAX) was developed by Hardman et al. to reveal human non-canonical phosphorylation. A total of 781 unique N-pho sites, including 225 pHis, 278 pLys, and 278 pArg, were identified from HeLa lysates Table , which m52. After re-examining our data, some vital N-pho proteins that perform protein kinases or phosphatases were identified, such as Phosphoenolpyruvate synthase and Phosphoenolpyruvate-protein phosphotransferase. Although several known pHis sites were successfully identified in our study, we failed to find some known p-His sites, including NME1/2 1-pHis118, NDK1 1-pHis 117, and PGAM 3-pHis11. The similar phenomenon was found in a previous report45. It deserved in-depth analysis, and there were three possible reasons for explaining failure to identification of known pHis sites: (i) The enrichment of N-pho is carried out under neutral conditions, and so negative charge peptides would be co-eluted with N-pho peptides in this case, which might inhibit N-pho peptides identification. (ii) Some tryptic pHis-containing peptides like the histone H4 (H18) and ACLY (H760) peptides are too short or too long to be considered by MS using current parameters29. (iii) Acid separation conditions would increase the risk of pHis hydrolysis29.pHis is physiologically important for both bacteria and mammals2@DpaZn: specific complexation with N-pho peptides driven by coordinate interaction at moderate binding ability, rapid adsorption within 10 min enabled by core–shell structure, high recovery and modification preservation achieved by on-tip analysis, as well as excellent enrichment ability toward diverse N-pho peptides  from biological samples. A large amount of unique N-pho sites have been identified from E. coli and HeLa lysates benefiting from these different characteristics from traditional TiO2 or IMAC materials. High discovery rates for N-pho sites and non-discrimination of recognizing all kinds of phosphorylated peptides illustrate the great potential of our material in comprehensive N-phosphoproteome analysis. Therefore, this universal method might greatly promote the studies of the world of N-phosphoproteins. In addition, the smart core–shell structure design and fast on-tip enrichment concept can be extended to other protein PTMs such as polyphosphorylation53 and S-sulphenylation54, which play vital roles in biological processes but are all fragile under certain conditions and cannot be efficiently captured by artificial materials so far. By precise designing the recognition groups, controlling the enrichment conditions, together with applying the fast on-tip enrichment, these challenges might be addressed. Furthermore, if the enrichment pH value was reduced to 2.3, SiO2@DpaZn may be suitable for large-scale pHis analysis. We believed that our core–shell structure design and fast on-tip enrichment concept would gain more attention in PTM proteomes analysis in the near future.In summary, the above results reveal four appealing features of SiO3·H2O), ammonium fluoride , 3- aminopropyltriethoxysilane , formaldehyde (37 wt% in water), sodium cyanoborohydride (NaBH3CN), sodium cyanoborodeuteride (NaBD3CN), trypsin (from bovine pancreas), protease inhibitor cocktail, formic acid (FA), urea, trifluoroacetic acid (TFA), dithiothreitol (DTT), iodoacetamide (IAA), and ammonium acetate were purchased from Sigma-Aldrich . Phosphatase inhibitor cocktail was ordered from Thermo Fisher Scientific . 13C-D2-formaldehyde was purchased from Cambridge Isotope Laboratories, Inc. . Dry pyridine was provided by Aladdin . Toluene and chloroform (≥99.0%) were bought from Kaixin , and toluene was purified by refluxing (110 °C) over sodium and distilling (136 °C). Acetone (≥99.5%) and ethanol (≥99.8%) were obtained from Kermel . Acetonitrile  was purchased from Merck . Deionized water was used throughout the experiments . Other reagents were all analytical grade. All the standard non-phosphorylated peptides were purchased from ChinaPeptides .Hexadecyltrimethylammonium chloride , tetraethyl orthosilicate , 1,6-diisocyanatohexane (≥99.0%), tridecane (≥99%), ammonium hydroxide solution  was measured by a bruker AVANCE III HD 400 MHz spectrometer at room temperature . Chemical shifts were recorded in parts per million (p.p.m.) using residual solvent peaks as internal references [CDCl3δ: 7.26 (1H)]. Molecular mass was detected by orbitrap LTQ . TEM images were collected on a transmission electron microscope operated at 120 kV . SEM images were collected on a Zeiss Merlin FEG-SEM instrument. TGA was performed under an air atmosphere with a heating rate of 10 °C/min using a Netzsch STA449F3 thermogravimetric analyzer . Zeta potential was measured using the Nano-ZS90 zetasizer . X-ray photoelectron spectroscopy (XPS) characterization was carried out on an Thermo ESCALAB250Xi spectrometer with Al Kα radiation as the X-ray source . The nitrogen adsorption and desorption isotherms were measured by QuadrasorbSI . The Brunauer–Emmett–Teller (BET) method was used to calculate the specific surface areas with adsorption data in a relative pressure range from 0.049 to 0.252. Contact angle was measured by DSA100 . MALDI-TOF spectra were recorded on Bruker Ultraflex III MALDI-TOF/TOF MS  and MALDI-TOF/TOF 5800 System  under positive reflection mode. ITC experiments were carried out on a MicroCal iTC200 instrument .Hydrogen NMR  and paraformaldehyde  solution (water/i-PrOH = 5:3 (v/v), 48 mL) was adjusted to pH 8.0 by 1 N HCl aq. After stirring at 80 °C for 30 min, Boc-L-tyrosine-OMe  was added, and the mixture was refluxed at 110 °C for 13 h. Then, the mixture was cooled to room temperature, and i-PrOH was removed by evaporation. After cooling on an ice-bath, the solution was removed by decantation, and the precipitated viscous oil was dissolved in 50 mL of AcOEt. The solution was washed with saturated NaHCO3 and brine followed by drying over Na2SO4. After removal of the solvent in vacuo, the residue was purified by column chromatography ) to give 1  as a yellow oil. 1H-NMR : δ 8.53 , 7.62 , 7.47 , 7.13 , 6.99 , 5.23 , 4.49–4.51 , 3.85 , 3.76 , 3.59–3.61, 2.99 , 1.35 . ESI-MS m/z for C41H47N7O5 [M+H]+ 718.35 and [M+Na]+ 740.20  on ice-bath, and the solution was stirred at room temperature for 2 h. After concentration in vacuo, the residue was dissolved in water and alkalized with ammonium hydroxide solution on ice-bath. The resulting mixture was extracted with CH2Cl2. The combined organic layers were washed with brine and dried over Na2SO4. After removal of the solvent in vacuo, 2  was obtained as a pale viscous oil. 1H-NMR : δ 8.53 , 7.61 , 7.46 , 7.13 , 7.03 , 3.89 , 3.81 , 3.75–3.70 , 3.68 , 3.01–2.96 , 2.80–2.75 . ESI-MS m/z for C41H45N7O6 [M+H]+ 618.48, [M+Na]+ 640.34  was added dropwise to a stirring solution of 1  in anhydrous CH2Cl2 (110 mL). The mixture was stirred and refluxed at 50 °C overnight. After removing the solvent by evaporation, 3 was obtained as a light yellow viscous oil. 1H-NMR : δ 8.53 , 7.61 , 7.46 , 7.13 , 7.03 , 3.86 , 3.79 , 3.70–3.65 , 3.01–2.96 , 2.80–2.75 . ESI-MS m/z for C41H45N7O6 [M+H]+ 732.53, [M+Na]+ 754.41  was added to a solution of 2  in anhydrous CH56. It contains three steps. Firstly, hydrolysis solution, containing 6.7 mL of NH3·H2O, 5.1 mL of H2O, and 70 mL of CH3CH2OH, was placed in water bath at 22 °C, and 4.0 mL of TEOS was added. The mixtures were reacted for 40 min under constant mechanical stirring. After the temperature was increased to 55 °C, 0.64 mL of H2O and 4.0 mL of pre-heated TEOS were added to the mixtures and reacted for 40 min (denoted as growth process), and the growth process was repeated another three times. The obtained suspension was denoted as seeds and divided into four pieces. Secondly, we used 1 piece seed replaced equal volume hydrolysis solution, and hydrolysis solution containing seed was heated to 55 °C, and the growth process was repeated three times. Afterward, the hydrolysis solution was added to the mixtures, and growth process was repeated four times. The obtained particles were centrifuged at 4000 × g for 5 min, with 95% (v/v%) CH3CH2OH and H2O. The obtained particles were divided to four pieces which were denoted as large particles. Finally, one piece of large particle was re-dispersed in the hydrolysis solution at 55 °C, and the growth process was repeated three times. Non-porous silica was obtained by centrifugation at 3300 × g for 3 min using 95% (v/v%) CH3CH2OH and H2O.Non-porous microspheres were prepared by a seed-growth approach2O, 5.8 mL of tridecane, and 1.0 g of CTAC. Then, 26 mg of NH4F and 6.0 mL of NH3·H2O were added and the mixtures were carried out for 24 h at 90 °C under stirring. The product was obtained by centrifugation at 3300 × g for 3 min. Finally, the produce was dried at 65 °C for 2 h and calcined with a Ceramic Fibre Muffle Furnace  at 550 °C for 6 h in air. The heating procedure was from 25 to 550 °C at a ramp rate of 1 °C/min. The pore size was enlarged by etching in 5 M HCl at 120 °C for 12 h.Firstly, 1.0 g of above non-porous silica with were added to the growth system which included 100 mL of Hg for 5 min and sequentially washed with toluene, CH3OH, and CH3COCH3.In a typical reaction, 0.6 g of sub-2-μm superficially porous silica particles and 2.21 mL of APTES were added to 30 mL of anhydrous toluene, and the mixtures were stirred and refluxed at 110 °C for 24 h. The obtained particles were centrifuged at 4000 × 2O , and DIEA  in dry DMF (21 mL) was stirred at room temperature for 3 days. After filtration, the particles were subjected to sequential washing with toluene, methanol, and acetone. Then, bis-Zn (GA-DpaTyr-OMe)-sub-2-μm superficially porous silica particles (570 mg) were dissolved in water/MeOH , and an water/MeOH solution  of Zn(NO3)2·6H2O  was added drop-wise to the silica particles suspension. After stirring at room temperature for 72 h, the obtained particles were centrifuged at 4000 × g for 5 min, and sequentially washed with H2O, CH3CH2OH, and acetone.In a typical reaction, a solution of carboxyl terminal Dpa 3 , amino-functionalized sub-2-μm superficially porous silica particles (600 mg), EDC , HOBt·H1H-NMR and 31P-NMR studies of DpaZn with pho-His were carried out on a Brucker ADVANCE III (500 MHz) at 25 °C. Samples were prepared using pho-His in D2O in the absence or presence of DpaZn. TSP was used as an internal standard, and the observed peaks were individually assigned by the COSY experiments. In 31P-NMR experiment, 85% H3PO4 in H2O was used as an external standard.pHYSIMAR) and 500 μg of BSA digests were mixed and dissolved in 200 μL of 80% ACN . After incubation with 200 μg of SiO2@DpaZn at room temperature for 20 min, total volume of 400 μL 80% ACN, 400 μL 65% ACN, and 400 μL 0.001% NH3·H2O comprised washing steps, respectively. Phosphopeptides were eluted with 100 μL of 0.1% NH3·H2O and analyzed by MALDI-TOF MS.Five micrograms of standard N-phosphopeptide  containing 10 μg of O-phosphopeptide GK8 at room temperature. After incubation for 2, 5, 10, 15, 20, and 30 min, respectively, 40 μL of the mixture was centrifuged, and the concentration of GK8 was measured by UV analysis. The adsorption capacity (Q) was calculated as follows: Q = (C0−C) V/m, where m is the mass (mg) of SiO2@DpaZn, V (mL) the volume of incubation, C0 (mg/mL) and C (mg/mL) the concentrations of GK8 in the initial solution and the supernatant after the adsorption, respectively. Three sets of parallel experiments were conducted simultaneously.A total of 0.4 mg of SiO2@DpaZn in 80% ACN  was packed into a 20-μL micropipette tip by centrifugation at 5400 × g for 10 min, and then equilibrated with 10 μL of 80% ACN . Sample loading, washing, and elution were realized by centrifugation at 5400 × g for 5, 12, and 5 min, respectively. Dissolved in 20 μL of 80% ACN, peptides were load onto SiO2@DpaZn tips. After washing three times with 80 μL of 50% ACN , 80 μL of 0.001% NH3·H2O, and 80 μL of 0.005% NH3·H2O orderly, samples were eluted with 10 μL of 1% NH3·H2O. Eluates were analyzed by MALDI-TOF MS.A total of 1 mg of SiOE. coli (strain K12) was cultured in Luria–Bertani medium  at 37 °C overnight or in M9 minimal medium  glucose or 0.5% glycerol) at 37 °C to exponential phase and stationary phase, and were harvested by centrifugation at 4000 × g for 2 min at 4 °C. Cells were washed three times with cold PBS and resuspended in lysis buffer  protease inhibitor cocktail, 10 mM EDTA, and 1% (v/v) phosphatase inhibitor cocktail). After ultrasonication for 120 s  in an ice bath, insoluble portions were separated from the soluble ones by centrifugation at 16,000 × g for 30 min at 4 °C, and protein concentrations were determined using BCA assay.E. coli protein extracts were denatured at 95 °C for 1 min. Then, the disulfide bond was reduced with 25 mM DTT at 37 °C for 75 min and the resulting cysteine residues were alkylated with 50 mM IAA at room temperature in the dark for 30 min. The excess IAA was quenched with 50 mM DTT. The resulting mixture was diluted with 20 mM ammonium bicarbonate to the final concentration of urea <1 M. Afterward, trypsin (1:3) was added and incubated at 37 °C for 1.5 h. The resultant peptides were desalted by RP-LC.2 and 95% air at 37 °C. All cells reaching 80% confluence were detached with 0.25% trypsin/EDTA and centrifugated  to collect the cells. Cells were washed three times with cold PBS and resuspended in lysis buffer  protease inhibitor cocktail, 10 mM EDTA, and 1% (v/v) phosphatase inhibitor cocktail). After ultrasonication for 120 s  in an ice bath, insoluble portions were separated from the soluble ones by centrifugation at 16,000 × g for 30 min at 4 °C, and protein concentrations were determined using BCA assay. After treating with DTT and IAA, the proteins were precipitated with CHCl3/CH3OH. Afterward, trypsin (1:3) was added and incubated at 37 °C for 1.5 h.The HeLa and HEPG2 cells were cultured in DMEM medium containing 10% FBS and 1% PBS in a humidified incubator of 5% CO2@DpaZn in 80% ACN was packed into 200-μL micropipette tips equally by centrifugation at 5400 × g for 10 min. Disolved in 200 μL of 80% ACN, 12 mg of E. coli digests were loaded onto SiO2@DpaZn tips by centrifugation at 5400 × g for 10 min. Washing steps were carried out with 100 μL of 50% ACN, 100 μL of 30% ACN, 100 μL of 0.001% NH3·H2O, and 80 μL of 0.005% NH3·H2O, respectively, and phosphopeptides were eluted with 20 μL of 1% NH3·H2O for two times. Elutes was mixed up for the following high pH-RP fractionation. For mammalian cell phosphopeptides, 30 mg of SiO2@DpaZn was used for 6 mg of cell lysate digests enrichment.A total of 36 mg of SiOOne microliter of the sample was deposited on the sample plate and dried, then 1 µL of 20 mg/mL DHB substrate (in 60% (v/v) acetonitrile and 0.1% (v/v) TFA aqueous solution) was introduced. MS spectra were acquired on an Ultraflex III TOF/TOF mass spectrometer  equipped with a 200-Hz smart-beam 1 laser at 355 nm or a MALDI-TOF/TOF 5800 System  quipped with a 1-kHz optibeam™ on-axis laser in the positive reflection mode, and controlled by the Flex Control 2.4 software and Data Explorer software 4.11. Flex Analysis software 3.3 and TOF/TOF Series Explorer software 4.1 were used to analyze MALDI-TOF MS data. The threshold for peak acceptance was a signal-to-noise ratio of 6.E. coli lysate digests analysis, each fraction of the enriched phosphopeptides was automatically loaded onto a RP trap column (150 μm i.d. × 5 cm) and separated by a C18 capillary column (150 μm i.d. × 15 cm). The trap column and the analytical column were both packed in-house with 5 μm, 100 Å Venusil XBP C18 silica particles. In order to study the phosphorylated peptides in E. coli cultured in LB medium, two mobile phases (A: 2% (v/v) ACN with 0.1% (v/v) FA and B: 98% (v/v) ACN with 0.1% (v/v) FA) were used to generate a 75-min gradient with the flow rate of 500 nL/min . A Q-Exactive mass spectrometer  was operated in full scan  and product scan  modes at positive ion mode. The electro-spray voltage was 2.3 kV, and the heated capillary temperature was 270 °C. All the mass spectra were recorded with Xcalibur software . MS/MS spectra were acquired by data-dependent acquisition mode, and the 20 most intense peaks with charge state ≥2 were selected for sequencing in the HCD collision cell with stepped collision energy of 28%, 30%, and 32%. Each fraction was analyzed in duplicate. Furthermore, in order to study the phosphorylated peptides in E. coli cultured in M9 medium, two mobile phases (A: 0.1% (v/v) FA in water and B: 98% (v/v) ACN with 0.1% (v/v) FA) were used to generate a 121-min gradient with the flow rate of 600 nL/min . A Orbitrap Fusion Lumos mass spectrometer  was operated in full scan  and product scan  modes at positive ion mode with orbitrap detector. The electro-spray voltage was 2.3 kV, and the heated capillary temperature was 320 °C. All the mass spectra were recorded with Xcalibur software . MS/MS spectra were acquired by data-dependent acquisition mode, and total cycle time was set to 3 s. Peptides with charge state ≥2 were selected for sequencing in the HCD collision cell with collision energy of 30%. HPLC separation for Hela lysate is as same as that for E. coli cultured in M9 medium, and an Orbitrap Fusion Lumos mass spectrometer  was operated in full scan  mode at positive ion mode with orbitrap detector. The electro-spray voltage was 2.5 kV, and the heated capillary temperature was 320 °C. All the mass spectra were recorded with Xcalibur software . MS/MS spectra were acquired by data-dependent acquisition mode, and total cycle time was set to 3 s. Peptides with charge state ≥2 were selected for sequencing with HCD  and neutral-loss-triggered (Δ97.98) EThcD  for fragmentation. All product ions were detected in the ion trap (rapid mode).For E. coli cultured in LB medium) with MASCOT 2.4 and Maxquant 1.6.0 (for the phosphorylated peptides in E. coli cultured in M9 medium and HeLa) were applied for database searching against the NCBI_E.coli_k12 database  or Uniprot_Homo Sapiens database . The corresponding reversed database was also performed to evaluate the false discovery rate (FDR) of peptide identification in the database searching process. The parameters of database searching included: up to three missed cleavages allowed for full tryptic digestion, precursor ion mass tolerance 10 p.p.m., product ion mass tolerance 20 m.m.u., carbamidomethylation (C) as a fixed modification, and oxidation (M), phosphorylation (STYHRK), acetyl (protein N-term), deamidated (NQ) as variable modifications. PSMs were validated using perculator based on q-value at a 1% FDR. GO analysis was carried out on http://pantherdb.org/ (Panther 15.0) and http://revigo.irb.hr/. Sequence motif analysis was carried out on Weblogo 2.8.2.Proteome Discoverer 2.1  by integration of the appropriate reaction peaks. The binding parameters  were evaluated by applying one site model using the software Origin .ITC titration was performed on an Isothermal Titration Calorimeter from MicroCal Inc. All measurements were conducted at 298 K. In general, a solution of the peptide (1–2 mM) in 50 mM HEPES buffer (pH 7.2) was injected stepwise (10 μL × 24 times) into a solution of DpaZn (25–100 μM) dissolved in the same solvent system. The measured heat flow was recorded as function of time and converted into enthalpies (ΔFurther information on research design is available in the Supplementary InformationPeer Review FileDescriptions of Additional Supplementary FilesSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Reporting Summary


Using the speed vs. lactate concentration curve, the speed at lactate threshold (sLT) and parameters corresponding to sLT were calculated . In the following day, a T30 corresponding to sLT was performed and BL-T30, VO2-T30, HR-T30, SR-T30, and SL-T30 were measured after the 10th and 30th minute, and average values were used for comparison. VO2-sLT was no different compared to VO2-T30 (p > 0.05). BL-T30, HR-T30, and SR-T30 were higher, while SL-T30 was lower compared to BL-sLT, HR-sLT, SR-sLT, and SL-sLT (p < 0.05). Continuous swimming at speed corresponding to lactate threshold may not show the same physiological and biomechanical responses as those calculated by a progressively increasing speed test of 7 × 200 m.The purpose of this study was to verify the physiological responses and biomechanical parameters measured during 30 min of continuous swimming (T30) at intensity corresponding to lactate threshold previously calculated by an intermittent progressively increasing speed test (7 × 200 m). Fourteen competitive swimmers (18.0 (2.5) years, 67.5 (8.8) kg, 174.5 (7.7) cm) performed a 7 × 200 m front crawl test. Blood lactate concentration (BL) and oxygen uptake (VO Progressive discontinuous swim protocols, such as a 7 × 200 m progressively increasing speed test, are commonly used to evaluate both physiological ,2 and biIn swimming, the most frequently used method for sLT calculation is x-axis projection of the intersection of two lines connecting the three higher and four lower points of the speed lactate curve . SubsequThus, the purpose of the current study was to verify the physiological responses and biomechanical parameters during continuous swimming at intensity corresponding to the lactate threshold previously calculated by an intermittent progressively increasing speed test (7 × 200 m). We hypothesized that the calculated parameters would be verified during the continuous swimming effort.Fourteen regional and national level male and female swimmers (ten sprinters and four middle-distance swimmers) that specialized in competitive distances of 100, 200, and 400 m volunteered to participate in the study . Partici−1) low volume endurance training (~3000 to 4000 m). The study was conducted during the specific preparation mesocycle of training. All swimming tests were performed using front crawl in a 25 m indoor swimming pool with a constant water temperature of 25 °C to 26 °C and 60% ambient humidity.Physiological and biomechanical parameters calculated during a progressively increasing speed swimming test  were compared to those measured during 30 min of continuous swimming. Swimmers were tested in two sessions 48 h apart , and all+, SensLab GmbH, Leipzig, Germany). Immediately after the completion of each 200 m repetition, a mouthpiece and a nose clip were attached to swimmers during recovery. Expired air collected during the first 20 s of recovery was analyzed for oxygen uptake (VO2) using a portable gas analyzer , mean r = 0.99 (0.01); 1). VO2-sLT and HR-sLT were calculated by interpolation using linear regression of swimming speed versus VO2 (mean R2 = 0.99 (0.02), mean r = 0.99 (0.01)) or HR (mean R2 = 0.99 (0.01), mean r = 0.99 (0.01). During the 7 × 200 m test, SR was calculated by the time (T) used to complete three arm-stroke cycles (180·T−1) and SL was calculated by dividing swimming speed every 50 m (V) by SR. The time for three arm-stroke cycles was recorded using a handheld chronometer. SR-sLT and SL-sLT were calculated by interpolation using the best fit regression line of SR and SL versus swimming speed during the 7 × 200 m test (SR: mean R2 = 0.98 (0.02), mean r = 0.99 (0.01); SL: mean R2 = 0.96 (0.04), mean r = 0.98 (0.02)).All swimmers participated in a standardized swimming warm-up, and 10 min later performed a 7 × 200 m front crawl test at intensities calculated using the most recent 200 m race time and corresponding to 60%, 70%, 75%, 80%, 85%, 90%, and maximum effort. Participants were familiarized with the pace of the first two repetitions during a previous training session. During testing, one of the experimenters walked alongside the swimming pool providing guidance during each 200 m repetition. During the 7 × 200 m test, each repetition started every five minutes and 30 s with a push-off start from within the water [N, USA.; ). HR was2-T30 were measured after the 10th and 30th minute of T30, while HR-T30 was recorded continuously. SR-T30 and SL-T30 were measured every 50 m during the T30 continuous swimming session. The 10th min was used to identify variations in physiological adjustments compared to the 30th min. Nevertheless, the mean values of BL, HR, SR, SL, and speed (s-T30) measured during the T30 were used for the statistical analysis. The swimmers participated in a continuous swimming session 48 h after the completion of the 7 × 200 m test. Continuous swimming speed session was completed after a standardized warm-up that consisted of 1000 m of swimming . Ten minutes after warm-up, the swimmers started a T30 at a constant speed corresponding to sLT. During T30, the swimmers kept the individual sLT speed constant while guided by a sound signal emitted by a transmitter placed next to the ear and under the swimming cap . The swimmers were instructed to adjust their speed in order to touch the wall at each sound signal. Additionally, one of the experimenters recorded the time for each 50 m split . BL-T30 and VOt-test for dependent samples was used to examine the differences in physiological and biomechanical parameters calculated during the 7 × 200 m progressively increasing speed test and those measured in T30. Specifically, comparisons between sLT, BL-sLT, VO2-sLT, HR-sLT, SR-sLT, SL-sLT vs. s-T30, BL-T30, VO2-T30, HR-T30, SR-T30, SL-T30, respectively, were applied. Pearson r correlation coefficient was used to examine the relationship between the calculated values from a 7 × 200 m test with that measured during T30. The effect size for paired comparisons using the pooled standard deviation as denominator was calculated with Cohen’s d [d was less than 0.20, small if it was between 0.20 and 0.50, medium if it was between 0.50 and 0.80, and large if it was greater than 0.80. The 95% confidence limits (95% CL) were also calculated for the mean differences between parameters. G-Power 3.1.9.4 software [N = 14), an ES of 0.80 was required to get a statistical power value greater than 0.80. For the estimation of agreement between parameters, Bland and Altman plots were used [p < 0.05.The student ohen’s d . The effsoftware  was usedere used . SPSS so−1, mean difference (SD): −0.002 (0.010) m·s−1, 95% CL: −0.008, 0.005·m·s−1, d = −0.02, p = 0.634). A significant correlation was observed between sLT and s-T30 .sLT calculated after the 7 × 200 m test was similar with s-T30 (sLT: 1.317 (0.078) vs. s-T30: 1.316 (0.082) m·s−1, mean difference (SD): 1.3 (2.4) mmol·L−1, 95% CL: 0.00, 2.35 mmol·L−1, d = 0.83, p = 0.05; R2 = 0.001, r = 0.03, p = 0.92). A Bland and Altman plot indicated agreement between calculated and measured values : 11 (11) b·min−1, 95% CL: 6, 18 b·min−1, d = 1.24, p = 0.02; R2 = 0.06, r = 0.25, p = 0.39). Agreement between calculated HR-sLT and measured HR-T30 values was observed (2-T30 was not different compared to VO2-sLT (VO2-T30: 41.7 (6.8) vs. VO2-sLT: 42.8 (6.2) ml·kg−1·min−1, mean difference (SD): −0.88 (4.5) ml·kg−1·min−1, 95% CL: −3.22, 1.45 ml·kg−1·min−1, d = −0.16, p = 0.43) and these parameters were correlated . A Bland and Altman plot showed agreement between calculated VO2-sLT and measured VO2-T30 values (Measured BL-T30 was higher compared to BL-sLT (BL-T30: 4.7 (2.3) vs. BL-sLT: 3.4 (0.8) mmol·Ld values . During observed . Moreove0 values .−1, mean difference (SD): 4.0 (3.9) cycles·min−1, 95% CL: 1.97, 6.07 cycles·min−1, d = 1.15, p = 0.002; R2 = 0.16, r = 0.41, p = 0.15). A Bland and Altman plot indicated agreement between calculated SR-sLT and measured SR-T30 values : −0.3 (0.2) m·cycles−1, 95% CL: −0.40, −0.14 m·cycles−1, d = −0.88, p = 0.001; R2 = 0.55, r = 0.74, p = 0.003). A Bland and Altman plot showed agreement between calculated SL-sLT and measured SL-T30 values  vs. SR-sLT: 29.7 (4.1) cycles·min0 values . Measure0 values .2 was similar to measured VO2, while a higher BL, HR, and SR and lower SL were recorded during T30. Bland and Altman plots indicated agreement, although a great bias was observed for all the physiological and biomechanical parameters.The purpose of the current study was to verify the physiological and biomechanical parameters measured during continuous constant speed swimming corresponding to lactate threshold with those calculated after a 7 × 200 m test. The calculated sLT was successfully maintained during a T30 session. Calculated VOA similar speed compared to sLT was expected in T30 on account of our experimental design. This is because swimmers were guided to follow a constant speed using audio signals. While maintaining the required speed during T30, some physiological adjustments were made by the swimmers. Increased BL and HR were observed during T30, and several factors may have contributed to this increment. First, we should examine the validity of sLT calculation with the method used in the present study. Previous studies report that using the intersection of two lines provides a speed corresponding to lactate threshold similar to MLSS . However−1 [A second factor that needs to be mentioned is that lactate threshold as well as MLSS may present lactate concentration at a range of 2 to 8 mmol∙L−1 . In such−1 . Support−1  found a −1 . Nonethe−1 . It seem−1 .−1) higher HR corresponding to lactate threshold using 400 m compared to 200 m stages [−1) was observed between HR-sLT and HR-T30, possibly attributed to the training status and specialty of the swimmers. Additionally, a likely HR drift towards the last minutes of exercise attributed to cardiovascular adjustments during the long exercise duration in T30 cannot be excluded [2-T30 was no different compared to VO2-sLT. It has been indicated that oxygen uptake reaches values between ~80–100% of VO2peak at the end of an endurance training set with a duration of 15 to 30 min [2 values that were ~85% of VO2max, similar to the current study (85.5%). A combination of steady VO2 response and increased BL during a continuous exercise may be observed during continuous efforts or in very heavy exercise intensity domains [A higher HR was observed during T30, indicating a higher effort during continuous swimming. A higher HR was measured in continuous exercise compared to that calculated corresponding to lactate threshold after a 7 × 200 m test in a previous study [excluded . In conto 30 min ,27. Spec domains .SR-T30 was increased whereas SL-T30 was decreased compared to SR-sLT and SL-sLT, respectively. Such changes are associated with an increased energy cost . It is p2-sLT, HR-sLT, SR-sLT, and SL-sLT and BL-T30, VO2-T30, HR-T30, SR-T30, and SL-T30, respectively, as indicated by Bland and Altman plots and has also been confirmed previously in a homogenous group of female middle- and long-distance swimmers [2-sLT, and HR-sLT as well as SR-sLT and SL-sLT were calculated by equations obtained by the best fit of these parameters versus swimming speed, thus reducing the error of calculation. However, this was not reflected in the measured values, indicating that the observed differences represent real physiological and biomechanical gaps between predicted and measured variables. Further research may examine various mathematical models for lactate threshold calculation in swimmers.The abovementioned differences in physiological and biomechanical parameters indicate that the calculated parameters may not always correspond to the measured values during continuous swimming. However, this is in contrast to the observed agreement between BL-sLT, VOswimmers . DespiteThe physiological and biomechanical parameters calculated by a progressively increasing swimming speed test corresponding to sLT may not be verified during continuous 30 min swimming in sprint and middle-distance swimmers. Swimmers maintaining constant speed corresponding to the second lactate threshold in a long duration 30 min swimming present individualized biomechanical and physiological adjustments that may not reflect the expected responses. In this case, an additional test for verification or a different mathematical model of lactate threshold calculation may be required to provide a valid guidance of training pace. Coaches should be aware that the individual data obtained by a progressively increasing speed test should be examined thoroughly and tested in training practice before planning a training set.


The structure of (1) was elucidated via single-crystal X-ray diffraction, mass spectrometry (MS), fourier transform infrared spectrometer (FTIR), and nuclear magnetic resonance (NMR) data analysis. Salumycin (1) contained a novel pyrazolequinone ring, which had never been previously reported in a natural product. Salumycin (1) exhibited moderate 2,2′-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging activity (EC50 = 46.3 ± 2.2 μM) compared with tert-butylhydroquinone (EC50 = 4.7 ± 0.3 μM). This study provides a new example of discovering novel natural products from bacteria.Heterocyclic natural products with various bioactivities play significant roles in pharmaceuticals. Here, we isolated a heterocyclic compound salumycin ( Bacteria are an important source of novel natural products and many bacterial natural products or their derivatives are currently used in human and animal pharmaceuticals, the food industry, and crop protection ,2. In reHeterocyclic natural products are unique compounds that have a wide range of biological, chemical, and physical characteristics, and more than half of small-molecule drugs contain a heterocyclic moiety, frequent examples are indole, pyridine, and thiazole as well as quinazoline rings ,13.Streptomyces albus J1074 for the discovery of new natural products. A S. albus mutant that could produce a new pyrazolequinone compound was obtained during the conjugation. Herein, identification of the S. albus mutant as well as the isolation, structure determination, and antioxidant activity of the new pyrazolequinone compound are reported.In our previous study, we constructed a metagenomic library derived from the environmental bacterial genomic DNA . We thenStreptomyces albus J1074 by conjugation  × 100Scavenging rate (%) = [1–{(A0 is the absorbance of the negative control (DPPH), A1 is the absorbance of the mixture (compound and DPPH), and A2 is the absorbance of the compound dissolved in MeOH.A1) as the GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor were calculated by using molinspiration online , according to the standard structure of the compound. The scores of more than 0.00, −0.50 to 0.00, and less than −0.50 corresponded to active, moderately active, and inactive bioactivities, respectively.The bioactivity scores of (8H7N3O2 (M = 177.17 g/mol): monoclinic, space group P21/c (no. 14), a = 4.8856 (3) Å, b = 10.0508 (7) Å, c = 16.1190 (12) Å, β = 95.743 (2)°, V = 787.54 (9) Å3, Z = 4, T = 296 (2) K, μ(MoKα) = 0.112 mm−1, Dcalc = 1.494 Mg/m3, 8627 reflections measured (5.08° ≤ 2θ ≤ 54.856°), 1786 unique , which were used in all calculations. The final R1 was 0.0422 (I > 2σ (I)) and wR2 was 0.1120.Crystal data for C1) was isolated from a S. albus J1074 mutant. Salumycin (1) contains a novel pyrazolequinone ring that has never been reported before in natural products. Salumycin (1) exhibited DPPH-radical scavenging activity, but no antibacterial activity. Genome analysis of the mutant showed a big genome deletion and exogenous DNA insertions. The biosynthetic mechanism of (1) needs to be further investigated and this study highlights again that bacteria are still an important resource for the discovery of novel natural products.In summary, a new compound salumycin (


Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs . Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity . Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral  A). Tat utilizes a nine-amino acid arginine-rich motif (ARM) to supplant an ARM-like counterpart contributed by the host protein HEXIM. Upon its release, pTEFb is escorted by Tat to the viral trans-activation response (TAR) element   . CDK9 wiFig. 1A) , 7. ThisThe precise role of arginine in Tat-mediated RNA recognition came into focus only recently based on the high-resolution NMR analysis of an HIV Tat-ARM in complex with human 7SK RNA and HIV TAR RNA . When boDK values in the nanomolar to picomolar range, while yielding iK values of ∼40 μm in antiviral assays . Starting from the high-affinity interaction between the U1A RNA recognition motif (RRM) and U1 small nuclear RNA (TAR-binding proteins (TBPs) was identified that bind TAR with nanomolar affinity (B) (i.e. TBP6.7), which revealed how three arginines within the lab-evolved β2-β3 loop recognize TAR at three conserved guanine nucleobases (DK of 1.8 ± 0.5 μm (B). Variations in the number and spacing of these arginines suggested the possibility that some variants use all four arginines to recognize TAR, that new modes of recognition—distinct from TBP6.7—were possible, and that some sequences and arginine spacings would be superior TAR binders compared with others.Recently a new class of arginine-dependent, protein-based TAR inhibitors was introduced that used a lab-evolution approach B. Startilear RNA , specifilear RNA , 27. Usiaffinity B 8, 27), 27B). Sleobases . This st± 0.5 μm . In cont± 0.5 μm  Fig. 1BBB. StartB) and a double mutant of TBP6.7 (Q48R/T50R) that contains five loop arginines. We then determined the corresponding co-crystal structures of these variants in complex with TAR. The results reveal how sequence differences in the β2-β3 loop cause structural changes that alter TAR recognition and stabilization of the β2-β3 loop. Based on these observations, we designed short macrocyclic peptides that mimic the β2-β3 loop and contain three or four arginines within a small (11-mer) cyclic peptide scaffold. Importantly, these compounds were determined to interact with TAR and to block Tat ARM binding to TAR, enabling the discovery of a new inhibitor of the TAR-Tat interaction. Our findings are discussed in the context of how diverse ARM interactions recognize RNAs and the principles of molecular recognition used by other macrocyclic peptides known to target the TAR-Tat interface.To address these questions and provide greater insight into TAR molecular recognition, we used isothermal titration calorimetry (ITC) to quantify how specific arginine compositions and spacings in the β2-β3 loop affect TAR binding. We evaluated TBP variants 6.3, 6.9, 6.6, 6.25 B and a dB) . T. TB) (28B) , and eac overfit . No brea overfit . The qua overfit  promptedA). This structure provides a benchmark for TAR-TBP–mediated interactions, which cluster in the TAR major groove and acquire specificity through Arg-47–, Arg-49–, and Arg-52–mediated readout of Gua26, Gua28, and Gua36 . Additional amino acids of the β2-β3 loop, such as Gln-48, recognize the phosphate backbone or form intrapeptide hydrogen bonds, such as Thr-50 and Gln-54, supporting a stable loop conformation (B and C).The co-crystal structure of HIV-1 TAR in complex with TBP6.7 was determined previously in our laboratory to 1.80 Å resolution . This woFig. 2ormation , B and CD). Conformational similarity was confirmed by the average r.m.s.d. of 0.43 ± 0.10 Å obtained from a pairwise superposition of all nonhydrogen atoms from each TBP upon TBP6.7. Like TBP6.7, the β2-β3 loop of each variant reaches far into the major groove (D). Despite changes in sequence, each loop exhibited a similar overall conformation as indicated by an average pairwise r.m.s.d. of 0.78 ± 0.36 Å (E), where differences are most notable at positions 49, 50, and 52. The latter r.m.s.d. is larger than the average coordinate error of 0.34 ± 0.07 Å measured from all structures  and HIV TAR exhibits significant conformational dynamics and flexibility in solution . For theB) (Generation of the TBP family entailed a lab-evolution process that yielded multiple proteins that bind TAR and show three or four arginines at distinct locations within the β2-β3 loop B 8). Th. ThB)  and a favorable enthalpic contribution that drives binding , and a binding reaction driven by enthalpy  (A–C) and 4A and Krel by >13-fold . ITC analysis showed a DK of 45.2 ± 3.4 nm for TBP6.3, representing a ΔΔG° of +1.2 kcal mol−1 compared with TBP6.7—signifying a less favorable binding equivalent to ∼2 hydrogen bonds (Krel of 8-fold) . The co-crystal structure of the TAR-TBP6.7 complex revealed that Gln-48 interacts with the TAR backbone, whereas Thr-50 stabilizes the β2-β3 loop by hydrogen bonding to Arg-52 (B). However, we observed that Arg-50 of TBP6.3 displaced Arg-52, usurping its role in Gua36 recognition. Arg-52 adopted a new conformation that protrudes into the TAR bulge to interact with the backbone at the pro-Rp oxygen of Uri23 (D and G). Thr-48 had no deleterious effects except that its side chain is too short to hydrogen-bond with the phosphate backbone as observed for Gln-48 in TBP6.7 (B and 3D).To examine the role of arginine placement in TAR recognition, we first considered TBP6.3, which differs from TBP6.7 by the change of Q48T and T50R B. ITC ano Arg-52  Fig. 2BBB. ITC an TBP6.7 B and 3D.B). This observation prompted us to investigate TBP6.9 because it is identical to TBP6.7, except that position 48 is replaced by arginine. Unexpectedly, ITC revealed that TBP6.9 bound to TAR with a DK of 3.0 ± 0.3 nm and ΔΔG° of −0.4 kcal mol−1, indicating a nearly equivalent interaction energy compared with TBP6.7 and a slightly more favorable Krel of 0.6 ).Although TBP6.3 contains four arginines, this variant exhibited 8.5-fold weaker binding than TBP6.7 , which hFig. 1l of 0.6 . As bothD and G) rather than N7 and the Gua28 phosphate—as observed in TBP6.9 (E and H). Differences between TBP6.3 and TBP6.9 underscore two important observations: (i) optimal arginine composition and placement supports TAR recognition through distinct sets of interactions with the Hoogsteen edges of guanine bases and the phosphate backbone, and (ii) intramolecular hydrogen bond interactions within the β2-β3 loop contribute peptide stability for the TAR major-groove region.Although TBP6.3 contains four arginines like TBP6.9, the former exhibits two suboptimal interactions. Namely, the Arg-50 guanidinium is not co-planar with the Gua36 base, yielding a weak inclined hydrogen bond interaction to the base Hoogsteen edge. Similarly, accommodation of Arg-50 in place of Arg-52 pulls nearby Arg-49 away from the phosphate backbone such that the Arg-49 guanidium group hydrogen-bonds to O6 and N7 of Gua28 , D and Gn TBP6.9 , E and HB). ITC revealed high-affinity TAR binding based on a DK of 4.2 ± 0.3 nm  and Figs. 2B and E and F) and Figs. 1B and 3D). As such, backbone stabilization interactions are suboptimal for this variant.To further test the latter observations, we analyzed variant TBP6.6, which possesses threonine at positions 48 and 50 B. ITC reG° of −0.2 kcal mol−1 is slightly more favorable compared with TBP6.7, and its ΔG° is equivalent to TBP6.9 (B).A main take-home message for TBP6.6 is that stabilization of the β2-β3 loop by threonine-mediated intramolecular hydrogen bonding is favorable. TBP6.6's ΔΔo TBP6.9 . These oo TBP6.9 B.B), use aromatic residues to recognize their single-stranded RNA targets , it seems plausible that Tyr-48 cannot engage in either interaction due to the bulk of the phenolic side chain. Unfortunately, our efforts to produce X-ray diffraction quality crystals of the TAR-TBP6.25 complex were unsuccessful. As such, we turned to our previous analysis in which we explored the role of position 48. We showed that the Q48A mutation in the context of TBP6.7 produced a ΔΔG° of +0.5 kcal mol−1 corresponding to a Krel of 2.2 . To probe this outcome, we integrated five arginines into the β2-β3 loop of TBP6.7 as the Q48R/T50R mutant. We rationalized that a β2-β3 loop with five arginines might rearrange its side chains in a manner similar to TBP6.3 to accommodate additional guanidinium groups in the major groove. ITC analysis revealed that the new variant recognizes TAR with DK of 27.8 ± 6.5 nm and a ΔΔG° of +0.9 kcal mol−1 . This variant makes contacts to TAR in a manner analogous to both TBP6.3 and TBP6.9. Specifically, Arg-52 interacts with the pro-Rp nonbridging phosphate oxygen of Uri23 when displaced by Arg-50—as in TBP6.3. Arg-48 interacts with the pro-Sp nonbridging phosphate oxygen of Gua36 as in TBP6.9. Arg-47, Arg-49, and Arg-50 retain their interactions with major-groove guanines as seen in TBP6.3 (C). No apparent backbone stabilization interactions form between β2-β3 loop residues.To understand the molecular basis of these thermodynamic properties, we next determined the co-crystal structure of the TAR-TBP6.7 Q48R/T50R complex. Again, the structure indicated that only three arginines could bind simultaneously to the Hoogsteen edges of guanine bases in the context of the β2-β3 loop B. This vn TBP6.3 C. No appAnalysis of the TBP6.7 Q48R/T50R double mutant highlights important aspects of TAR recognition. First, the addition of extra arginines in the β2-β3 loop—beyond the initial three—did not promote additional interactions at the Hoogsteen edges of guanine bases. Second, the need to maintain charge separation appears to have influenced arginine locations and rotamer conformations in the β2-β3 loop. Third, residues that fill the gaps between base-recognition arginines were of benefit when they stabilized the polypeptide via intramolecular hydrogen bonds.i.e. TAR-binding cyclic peptide from TBP6.7a in which appended letter “a” represents a methylene linker). The peptide encompasses the 46PRQRTPRGQ54 sequence corresponding to the β2-β3 loop of TBP6.7 (i.e. TB-LP-6.7) was also prepared in which the cysteines were alkylated with iodoacetamide . Importantly, cyclic peptide TB-CP-6.7a was found to interact with TAR with a kon of 7650 ± 190 m−1 s−1 and a koff of 0.028 ± 0.004 s−1, corresponding to a DK of 3.6 ± 0.4 μm . As a control experiment, we first titrated the Tat ARM into TAR RNA. The interaction produced a strong heat of binding that gave a DK of 135.0 ± 31.0 nm , demonstrating that TB-CP-6.9a formed a complex with TAR that occluded Tat-binding sites.We used an ITC-based competition analysis derived from a previously reported assay  wherein Fig. 6d by SPR . We nextC), this peptide showed negligible heats of binding when titrated into TAR  ; (iii) a bulky aromatic group at position 48 was not well-tolerated (e.g. TBP6.25); and (iv) Arg-50 displaced Arg-52, causing suboptimal Gua36 recognition (e.g. TBP6.3 and TBP6.7 Q48R/T50R). Collectively, these observations illustrate why lab-evolved β2-β3 loops obey the known consensus sequence  while providing a rationale for the use of specific sequences in the synthesis of small cyclic peptides that target TAR.HIV/AIDS is a pernicious public health threat with no vaccine or cure that has exacerbated other medical emergencies . As viraFig. 1A) . On the B and C), D–I), and B and C)). Given this observation, we sought to identify whether other RNA-binding proteins showed similar limitations. A brief survey of relevant RNA-protein interactions featuring major-groove recognition by ARM elements revealed a parallel trend in arginine-mediated interactions. To illustrate, we considered the fragile-X mental retardation protein (FMRP) bound to G-quadraplex RNA . In another compelling example, the Csy4-crRNA complex reveals that the Csy4 endoribonuclease domain contains an α-helical arginine-rich motif that uses only one arginine in a cluster of six to make base-specific interactions  or make no RNA contacts whatsoever.A significant discovery from our co-crystal structures was that only three arginines from each β2-β3 loop could be used simultaneously for guanine recognition  and E, F, H, and I). Additionally, Lys-50 and Lys-51 of Tat make phosphate-specific backbone interactions with TAR, whereas Arg-55 interacts with the backbone of 7SK. Such contacts are similar to Arg-52 of TBP6.3 and Arg-48 of TBP6.9 .Use of distinct islands of arginines within a single ARM to recognize different RNA targets is exemplified by recent solution structures of HIV Tat in complex with human 7SK RNA and HIV TAR RNA . The TatFig. 7Recognition of different RNAs by a single ARM that adopts different conformations has been dubbed “chameleonism” by Frankel . The HIVD and G) and B and C)). Comparable plasticity has been reported elsewhere for various arginine-rich motifs  and bona fide antiviral activity in cell culture (B and C) and D–I)). Unlike the TBPs, however, JB181 does not use amino acid side chains to engage in intrapeptide stabilization, such as Thr-48 and Thr-50 of TBP6.6 (F and I) and B and C).To better understand the structural features needed for TAR targeting by short peptides, it is also instructive to compare the TAR-binding features of TBPs herein with those of JB181. The latter β-hairpin peptide was derived from BIV-TAR and has undergone multiple cycles of optimization to yield ultrahigh affinity for TAR ). Simultaneously, Nε and Nη2 of Arg-47 hydrogen-bond to the pro-Rp and O5′ oxygens within the Uri23 phosphate backbone, thus giving a two-pronged “fork” readout; the Arg-47 guanidinium is flanked by cation-π contacts to Ade22 and Uri23 that further strengthen the mode of binding. The recent HIV-1 Tat-TAR complex reveals an analogous interaction between Arg-52 and Gua26 (C). Significantly, methylation analysis and mutagenesis of each arginine in the arginine-rich motif of HIV Tat revealed that Arg-52 alone is sufficient for TAR binding and trans-activation  cyclic peptide, TB-CP-6.9a, that is able to interact with TAR with a comparable DK of 3.6 ± 0.4 μm  of their arginine-rich sequences, which likely undermines their ability to recognize the target RNA with high affinity and/or specificity.Although JB181 was generated by structure-based design, our investigation herein evaluated the feasibility of generating small TAR-binding cyclic peptides derived from the lab-evolved β2-β3 loops of TBPs. Previously, we reported a 20-mer cyclic peptide, peptide 1  that comprises the entire β2-β3 strand-loop-strand element of TBP6.7 cyclized via a perfluoroaryl linker . Peptide± 0.4 μm . This re± 0.4 μm , thus re± 0.4 μm . This reversus TB-CP-6.7a is mainly driven by an increase in association rate constant (kon) for the interaction with TAR . Such knowledge was instrumental for the development of a small cyclic peptide mimic of the β2-β3 loop that is able to bind TAR and inhibit its interaction with the Tat ARM. Whereas further optimization of this compound is warranted, this work paves the way to the development of a new class of inhibitors of the TAR-Tat interaction. It also demonstrates the feasibility of using compact cyclic peptides to target viral RNAs—a subject of great concern given the ongoing coronavirus pandemic ; in addition, the Y31H/Q36R mutant was integrated into each variant to promote crystallization (Escherichia coli BL21(DE3) (NEB Inc.). Freshly streaked colonies grown on Luria–Bertani (LB) agar plates under selection conditions at 37 °C were used to inoculate overnight LB liquid cultures containing 50 μg/ml kanamycin. The overnight cultures were used to start large-scale LB growths at 37 °C that were induced during mid-log phase by 0.5 mm isopropyl 1-thio-β-d-galactopyranoside added to the media. Cells were harvested after 4 h of growth at 20 °C, and pellets were frozen in liquid N2. Cells were thawed in a cell lysis buffer (CLB) containing 0.05 m Na-HEPES, pH 7.5, 0.5 m NaCl, 0.02 m imidazole, pH 8.0, 0.0005 m EDTA, 0.005 m β-mercaptoethanol (β-ME) and 0.01% (v/v) Brij35; the cell slurry was made 2 mg ml−1 in lysozyme (VWR). After 20 min, cells were sonicated, and the clarified supernatant was applied in batch to nickel-nitrilotriacetic acid resin (Pierce) equilibrated with CLB. After 2 h of nutation at 4 °C, resin was poured into a 1.5 × 10-cm gravity-flow column , washed with 40 column volumes of CLB and 2 column volumes of wash buffer containing 0.05 m Na-HEPES, pH 7.0, 0.3 m NaCl, 0.04 m imidazole, pH 7.5, 0.005 m EDTA, 0.005 m β-ME, and 0.01% (v/v) Brij35. Elution was in 3-ml fractions using an elution buffer containing 0.05 m Na-HEPES, pH 7.0, 0.15 m NaCl, 0.2 m imidazole, pH 7.5, 0.005 m EDTA, 0.005 m β-ME, and 0.01% (v/v) Brij35. Fractions were pooled based on absorption at 280 nm and diluted with wash buffer to a final imidazole concentration <0.02 m. TEV  using an ÄKTA Pure (GE Lifesciences). The sample was washed and eluted using a linear gradient comprising 0.15–0.85 m NaCl, 0.05 m Na-HEPES, pH 7.0, 0.0025 m EDTA, and 0.00025 m β-ME. TBP6.3 and TBP6.9 were purified similarly except that a 0.15–1.5 m NaCl salt gradient was used for the HiTrap SP FF column to reduce nonspecific RNA interactions. Each concentrated protein sample was gel-filtered using a HiPrep (16/60) Sephacryl S-300 HR column (Cytiva). TBPs (11.5 kDa) exhibited higher retention than predicted by mass, eluting at or >1 column volume. The yield was 2–3 mg/liter of cells.TBP6.3, TBP6.6, TBP6.7, TBP6.7 Q48R/T50R, TBP6.6, and TBP6.25 DNA were each prepared from a synthetic gene cloned into pUC57 (GeneScript Inc.). Variants were derived from the human U1A protein sequence modified to produce sequences generated from yeast display , 66 FigB; in addlization , 66. TBP2 m. TEV  was addeC) was generated by chemical synthesis (Horizon Discovery) and purified as described  and incubated at 65 °C for 2 min. The RNA was cooled overnight to room temperature. TBP was titrated dropwise with gentle vortexing into a microcentrifuge tube containing the folded RNA to achieve a 1.2:1 molar ratio . The mixture was incubated at room temperature for 0.5 h and concentrated using a Nanosep 3K Omega spin-filter (PALL) to 10–12 mg ml−1 based on absorption at 280 nm. The final complex was 0.2-μm-filtered  and subjected to hanging-drop broad screens using a MosquitoTM robot (SPT Labtech). An equal volume of well solution was added to 0.2 μl of TAR-TBP complex. Most crystals appeared in 4–7 days at 20 °C as thin plates with a half-octagon habit. Crystals were typically 0.15 × 0.07 × 0.04 mm in size. TAR-TBP6.7 Q48R/T50R crystals grew directly from the JCSG-Plus screen (Jena Bioscience) from a well solution comprising 20% (w/v) PEG3350 and 0.2 m (NH4)2NO3. TBP6.9-TAR crystals grew directly from a Natrix broad screen (Hampton Research) from a well solution of 17.5% (w/v) PEG4000, 0.005 m MgCl2·6H2O, and 0.05 m MES monohydrate, pH 6.0. Other crystals were optimized from broad screens and were prepared by hanging-drop vapor diffusion using VDX plates (Hampton Research). For TBP6.3, an equal volume of well solution comprising 17.5% (w/v) PEG3350, 0.1 m Na-cacodylate, pH 6.0, and 0.2 m NH4HCO2 was added to 2 μl of TAR-TBP6.3 complex with equilibration over 1 ml of mother liquor at 20 °C. TAR-TBP6.6 crystals grew from 17% (w/v) PEG5000 MME, 0.1 m NaCl, 0.002 m (NH4)2SO4, and 0.05 m Na-cacodylate, pH 7.0. Cryo-protection of all crystals was by serial transfer of crystals into mother liquors supplemented in five steps from 5 to 20% (v/v) glycerol followed by snap cooling in liquid N2. X-ray data were recorded at the Stanford Synchrotron Radiation Lightsource. Data reduction statistics are shown in The HIV TAR 27-mer RNA C was genescribed . The RNAhttps://smb.slac.stanford.edu/facilities/software/xds/#autoxds_script), which employed the XDS and CCP4 packages  was pipetted into the RNA, followed by 2 min of incubation at 65 °C; the sample was cooled overnight to room temperature. Each sample was dialyzed overnight at 4 °C against 4 liters of ITC buffer. Following co-dialysis with RNA, protein samples were diluted in dialysis buffer to concentrations ∼10-fold higher than RNA. ITC measurements were conducted using a PEAQ-ITC  with protein in the syringe and RNA in the cell . Analytical HPLC was performed on a Shimadzu (LC-2010A HT) using an analytical C18 column  at a flow rate of 1 ml/min. Semipreparative HPLC was performed on an Agilent 1200 using a Hypersil GOLD C18 semipreparative column (10 × 250 mm) at a flow rate of 2.5 ml/minThe synthesis was carried out using Fmoc–solid-phase peptide synthesis on Knorr amide resin . Peptide synthesis was performed manually in syringes in the presence of 0.4 mmol amino acid (4 eq), 0.4 mmol HATU (4 eq), and 0.8 mmol DIPEA (8 eq). Initially, preswollen resin was treated with 20% piperidine in DMF containing 50 mmol of hydroxybenzotriazole (3 cycles 3-5-3 min) to remove the Fmoc-protecting group. Amino acids were coupled using the aforementioned reagent conditions. After coupling of the final amino acid, the Fmoc group was removed. The free amine at the N terminus was acetylated using 10 eq of acetic anhydride and 20 eq of DIPEA in DMF.The polypeptide product was cleaved from resin as follows: the resin was washed with DMF-DCM and dried by vacuum; the product was cleaved using a mixture of TFA/triisopropylsilane/water (95:2.5:2.5) for 2 h at room temperature; the cleavage mixture was filtered, and the combined filtrate was added dropwise to a 10-fold volume of cold diethyl ether and centrifuged; the supernatant was removed, and the precipitated crude peptide was dissolved in 10 ml of acetonitrile/water (1:1) and lyophilized.m ammonium bicarbonate buffer in 50% acetonitrile, 50% acetonitrile/ammonium bicarbonate buffer, pH 8.5. Tris(2-carboxyethyl)phosphine (TCEP)  was added to the peptide solution and incubated at 37 °C for 1 h. The diiodo-methane linker  was dissolved in DMF and added to the peptide solution and allowed to incubate at 37 °C for another 12 h. Progress of the reaction was monitored by MALDI-MS.Peptide cyclization was performed as follows: the lyophilized peptide (4 mmol) was dissolved in 20 ml of 0.05 After cyclization, the solution was lyophilized. The cyclic peptide was purified by semi-preparative HPLC using a gradient of 20–75% Buffer B in 30 min, which resulted an average overall yield of 10–12%. Representative MS data and HPLC traces for each peptide are provided in m HEPES, pH 7.5, and 0.15 m NaCl. A 200 nm stock of biotinylated TAR dissolved in SPR buffer was flowed over the chip surface to achieve 1200 RU. Analysis of TB-CP-6.7a proceeded in SPR buffer. To reduce nonspecific binding during TB-CP-6.9a assays, a 100-fold molar excess of yeast tRNA was added to SPR buffer. The flow rate for kinetics experiments was 75 μl/min. Cyclic peptides at various concentrations (10–160 μm for TB-CP-6.7a and 0.625–20 μm for TB-CP-6.9a) were injected for 60 s and allowed to dissociate for 180 s. To regenerate the RNA, 2 m NaCl was injected for 60 s. Experimental data were processed using the double-referencing method (kon and koff) and the apparent equilibrium binding constant (DK) (DK for the equilibrium binding measurements on TB-LP-6.9 was determined by taking the average response from a 5-s window at equilibrium (Req) for each peptide injection and plotting versus the peptide concentration using Prism software; data were then fit to a one-site binding model. The kinetic and equilibrium experiments were repeated in duplicate.Biotinylated-TAR 27-mer was deprotected and desalted by the manufacturer (Horizon Discovery). The RNA was folded as described for ITC (above). The RNA was immobilized on CM5 chips (Cytiva) conjugated to Neutravidin using an SPR buffer comprising 0.010 g method . The bufant (KD) . The resm Na-HEPES, pH 7.5, 0.05 m NaCl, 0.05 m KCl, and 0.002 m β-ME). The Tat ARM sequence  complex retained in the sample cell from the prior experiment. For the competition assay with linear peptide, 500 μm TB-LP-6.9 was titrated into 20 μm TAR in a dropwise manner and incubated on the bench at room temperature for 1 h. The Tat ARM at a concentration of 400 μm was then titrated into the preformed complex in the sample cell. This titration was best fit to a two-site binding model. All curves were fit using MicroCal PEAQ-ITC Analysis software . Average thermodynamic parameters are provided in For competition experiments, TAR RNA was folded as described (above) and dialyzed overnight against 4 liters of ITC buffer (0.05 sequence  was GISY binding . For the6XH0, 6XH1, 6XH2, and 6XH3.Coordinates and structure factor amplitudes have been deposited into the Protein Data Bank as entries


Cheiloneurusnankingensissp. nov., from Eastern China is described. It is similar to C.arabiacus Hayat but distinct from it in a number of morphological characters. It is a hyperparasitoid with the encyrtid wasp Aenasiusarizonensis Girault, 1915 as the primary host and the cotton mealybug Phenacoccussolenopsis Tinsley, 1898 (Hemiptera: Pseudococcidae) as the secondary host. A key to all seven species of Cheiloneurus known from China is presented.A new species,  Cheiloneurus (Encyrtidae) has been recorded as a hyperparasitoid of the cotton mealybug Phenacoccussolenopsis Tinsley, 1898  in North America  Moench. Laboratory observation showed that C.nankingensis adults produce offspring when attacking the mealybugs that had been parasitized by Aenasiusarizonensis Girault, 1915, but failed to do so when confined with healthy mealybugs, indicating that A.arizonensis is the host. This host parasitoid, having been synonymized with A.bambawalei Hayat, 2009, belongs to the subfamily Tetracneminae in Encyrtidae ; hind margin of scutellum reaching base of propodeum; mesopleura smooth, reaching base of abdomen; propodeum smooth.Fore wings: 3.01 × as long as broad, with uniform cilia except basal third; submarginal vein with about five setae; submarginal vein 3.90 × as long as stigmal vein; linea calva closed anteriorly by two or three lines of setae and posteriorly closed by 11 or 12 lines of hyaline setae.Legs: a row of spines at apex of mid tibia; tibial spur 0.96 × as long asbasitarsus ; basitarsus longer than tarsal segments 2–4.Metasoma: oblong in dorsal view. Ovipositor sheaths yellow. Exserted part of ovipositor sheaths (from slide) 0.15 × gaster length.Measurements (from slide): length of mid tibia 0.65 mm, mesosoma 0.73 mm, metasoma 0.70 mm, forewing 1.25 mm.Male. Length 1.07 mm. Body black. Antennae slender with radicle, scape and pedicel dark yellowish brown; funicle and clava black. Oval compound eye and ocelli black. Legs yellowish brown.POL and OOL 3.00 × and 0.92 × as long as diameter of anterior ocellus, respectively; toruli separated by 0.83 × their own longest diameters; mandible bidentate; toruli diameters 0.69 × and 2.26 × as long as POL and OOL, respectively; distance between compound eyes 3.45 × as long as that between toruli.Head: in frontal view, approximately triangular with scaly reticulations, 0.97 × as broad as high; Antennae: scape cylindrical, 6.53 × and 0.46 × as long as broad and head height, respectively; pedicel triangular; clava unsegmented, longer than F5–F6 combined; scape 3.35, 1.62, 1.95, 2.13, 2.14, 2.15, 2.18, and 1.05 times as long as pedicel, F1–F6, and clava, respectively; F1 slightly longer than F2–F6 separately; F2–F6 nearly same length; ratio of length to width 2.57, 2.20, 2.30, 2.22, 2.15, and 2.18 for F1 to F6, respectively.Mesosoma: In dorsal view, 1.35 × as long as width; mesoscutum, axillae, and scutellum with sculpture scaly and similar to head sculpture; scutellum 0.97 × as long as height; setae and notauli virtually absent.Fore wings: 2.42 × as long as broad; submarginal vein with about 13 setae; postmarginal and submarginal veins 0.58 × and 10.65 × as long as stigmal vein.Legs: mid tibia with row of spines apically; spur 1.08 × as long as basitarsus.Metanotum: nearly triangular in dorsal view. Shorter than thorax.Measurements (from slide): mesosoma length 0.55 mm, metasoma length 0.43 mm, antennae 0.86 mm, and mid tibia length 0.43 mm.C.arabiacus Hayat, (2014) but differs from it in the following respects (compared with C.arabiacus in brackets): without an infuscate area below proximal half of parastigma (with it); scape cylindrical (slightly expanded in the middle); pedicel as long as F1 (longer than F1–F2 combined); F3 quadrate or slightly broader than long (F3 longer than broad); F4–F6 broader than long ; clava shorter than F4–F6 combined (longer than F4–F6 combined); head in frontal view higher than broad (broader than high); mandible with three acute teeth (with two acute and one round teeth).This new species resembles A.arizonensis as the host, which is a primary and solitary parasitoid of the cotton mealybug P.solenopsis. This hyperparasitoid attacks only mealybugs that have already been parasitized by A.arizonensis and the number of offspring hyperparasitoids ermerging from a mummified mealybug ranges from one to 18. The prevalence of hyperparasitism by this wasp across the range of the cotton mealybug remains to be investigated.Little has been known about biology of this new species. It is a hyperparasitoid with the encyrtid wasp