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LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2–4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2–4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2–4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2–4, with long and short linkers, forms heterogeneous complexes, whereas QT4–6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed. [ABSTRACT FROM AUTHOR]

• Three novel linear PTO-based COPs was fabricated via Suzuki coupling reaction. • The P(PTODB)-2 shows a high initial specific capacity of 210 mA h g-1 at 0.1 A g-1. • Superior rate properties (150 mA h g-1 at 5 A g-1) could be achieved. • The ex-situ FT-IR and XPS tests have proved lithium storage mechanism with C=O groups. Conjugated organic polymers (COPs) have been emerged as a class of cathode materials for lithium-ion batteries (LIBs) because of the rigid structural units and tunable properties. Nonetheless, their applications are still limited due to the poor conductivity and low cycling life. Herein, we design a series of pyrene-4,5,9,10-tetraone-based COPs (P(PTODB)-1, P(PTODB)-2, P(PTODB)-3), containing different number of benzene rings as linking units. Consequently, prolonging the linker lengths could effectively improve structural stability and extend π-conjugation, leading to the enhanced charge-storage capability. When tested as cathode materials for LIBs, the P(PTODB)-2 electrode delivers a better electrochemical performance with high capacity of 203 mA h g-1 after 100 cycles at 0.1 A g-1 (coulombic efficiency almost 100%) and excellent rate performance (150 mA h g-1 at 5 A g-1). In addition, the Li+ storage mechanism was carried out by ex situ Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis. Eventually, our study brings forward the appropriately extended linker lengths to fabricate COP cathodes with high electrochemical properties. [Display omitted] [ABSTRACT FROM AUTHOR]

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects. [Display omitted] • Expanded toolset for swapping histones in S. cerevisiae • Complementation tests between human replicative histones and their variants • Amino acid swapping experiments between replicative human H2A and macroH2A1.2 • Human macroH2A1.2 alters the structure of yeast chromatin fiber locally and at long range Haase et al. used a genetic swapping assay to replace replicative human histones with non-replicative variants in yeast. H2A.J, TsH2B, and H3.5 complemented their replicative counterparts, while macroH2A1.2 proved toxic, disrupting chromatin organization, increasing nucleosome repeat length, and—when associated with chromosome instability—leading to fitness defects in yeast. [ABSTRACT FROM AUTHOR]

Zaibing Li,1,2,* Jingyu Li,1,* Liyan Liu,1 Wenyi Deng,3 Qingrong Liu,4 Ruofan Liu,4 Wen Zhang,3 Zaiqing He,5 Lei Fan,6 Yingzhuo Yang,7 Yun Duan,7 Huifang Hou,1 Xinyuan Wang,1 Zhimei Yang,1 Xiaoying Wang,1 Shanze Chen,1 Yi Wang,1 Ning Huang,1 Junli Chen1 1Department of Pathophysiology, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, People’s Republic of China; 2Department of Pathophysiology, School of Basic Medical Science, Southwest Medical University, Luzhou, Sichuan 646000, People’s Republic of China; 3West China Medical School, Sichuan University, Chengdu 610041, People’s Republic of China; 4West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, People’s Republic of China; 5Department of Pathology, Nuclear of Industry 416 Hospital, Chengdu, Sichuan 610051, People’s Republic of China; 6Department of Occupational Medicine, Nuclear of Industry 416 Hospital, Chengdu, Sichuan 610051, People’s Republic of China; 7Department of Nuclear Medicine, Sichuan Cancer Hospital, Chengdu 610041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Junli Chen; Ning Huang Email chenjunlinsf@163.com; ninghuangcjl@163.comBackground: α2A-adrenoceptor (AR) is a potential target for the treatment of degenerative diseases of the central nervous system, and α2A-AR agonists are effective drugs for this condition. However, the lack of high selectivity for α2A-AR subtype of traditional drugs greatly limits their clinic usage.Methods: A series of homobivalent 4-aminoquinolines conjugated by two 4-aminoquinoline moieties via varying alkane linker length (C2-C12) were characterized for their affinities for each α2-AR subtype. Subsequently, docking, molecular dynamics and mutagenesis were applied to uncover the molecular mechanism.Results: Most 4-aminoquinolines (4-aminoquinoline monomer, C2-C6, C8-C10) were selective for α2A-AR over α2B- and α2C-ARs. Besides, the affinities are of similar linker length-dependence for each α2-AR subtype. Among all the compounds tested, C10 has the highest affinity for α2A-AR (pKi=− 7.45± 0.62), which is 12-fold and 60-fold selective over α2B-AR and α2C-AR, respectively. Docking and molecular dynamics suggest that C10 simultaneously interacts with orthosteric and “allosteric” sites of the α2A-AR. The mutation of F205 decreases the affinity by 2-fold. The potential allosteric residues include S90, N93, E94 and W99.Conclusion: The specificity of C10 for the α2A-AR and the potential orthosteric and allosteric binding sites proposed in this study provide valuable guidance for the development of novel α2A-AR subtype selective compounds.Keywords: alpha2-adrenoceptor, selectivity, linker length, allosteric modulation

LC8, a ubiquitous and highly conserved hub protein, binds over 100 proteins involved in numerous cellular functions, including cell death, signaling, tumor suppression, and viral infection. LC8 binds intrinsically disordered proteins (IDPs), and although several of these contain multiple LC8 binding motifs, the effects of multivalency on complex formation are unclear. Drosophila ASCIZ has seven motifs that vary in sequence and inter-motif linker lengths, especially within subdomain QT2–4 containing the second, third, and fourth LC8 motifs. Using isothermal-titration calorimetry, analytical-ultracentrifugation, and native mass-spectrometry of QT2–4 variants, with methodically deactivated motifs, we show that inter-motif spacing and specific motif sequences combine to control binding affinity and compositional heterogeneity of multivalent duplexes. A short linker separating strong and weak motifs results in stable duplexes but forms off-register structures at high LC8 concentrations. Contrastingly, long linkers engender lower cooperativity and heterogeneous complexation at low LC8 concentrations. Accordingly, two-mers, rather than the expected three-mers, dominate negative-stain electron-microscopy images of QT2–4. Comparing variants containing weak-strong and strong-strong motif combinations demonstrates sequence also regulates IDP/LC8 assembly. The observed trends persist for trivalent ASCIZ subdomains: QT2–4, with long and short linkers, forms heterogeneous complexes, whereas QT4–6, with similar mid-length linkers, forms homogeneous complexes. Implications of linker length variations for function are discussed.

Detailed data from statistical analyses of the structural properties of the inter-domain linker peptides of the bacterial regulators of the family MocR are herein reported. MocR regulators are a recently discovered subfamily of bacterial regulators possessing an N-terminal domain, 60 residue long on average, folded as the winged-helix-turn-helix architecture responsible for DNA recognition and binding, and a large C-terminal domain (350 residue on average) that belongs to the fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes such aspartate aminotransferase. Data show the distribution of several structural characteristics of the linkers taken from bacterial species from five different phyla, namely Actinobacteria, Alpha-, Beta-, Gammaproteobacteria and Firmicutes. Interpretation and discussion of reported data refer to the article “Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators” (T. Milano, S. Angelaccio, A. Tramonti, M. L. Di Salvo, R. Contestabile, S. Pascarella, 2016) [1].

Liposomes (LPs) as commonly used mRNA delivery systems remain to be rationally designed and optimized to ameliorate the antigen expression of mRNA vaccine in dendritic cells (DCs). In this study, we synthesized mannose-cholesterol conjugates (MPn-CHs) by click reaction using different PEG units (PEG100, PEG1000, and PEG2000) as linker molecules. MPn-CHs were fully characterized and subsequently used to prepare DC-targeting liposomes (MPn-LPs) by a thin-film dispersion method. MPn-LPs loaded with mRNA (MPn-LPX) were finally prepared by a simple self-assembly method. MPn-LPX displayed bigger diameter (about 135 nm) and lower zeta potential (about 40 mV) compared to MPn-LPs. The in vitro transfection experiment on DC2.4 cells demonstrated that the PEG length of mannose derivatives had significant effect on the expression of GFP-encoding mRNA. MP1000-LPX containing MP1000-CH can achieve the highest transfection efficiency (52.09 ± 4.85%), which was significantly superior to the commercial transfection reagent Lipo 3K (11.47 ± 2.31%). The optimal DC-targeting MP1000-LPX showed an average size of 132.93 ± 4.93 nm and zeta potential of 37.93 ± 2.95 mV with nearly spherical shape. Moreover, MP1000-LPX can protect mRNA against degradation in serum with high efficacy. The uptake study indicated that MP1000-LPX enhanced mRNA expression mainly through the over-expressing mannose receptor (CD206) on the surface of DCs. In conclusion, mannose modified LPs might be a potential DC-targeting delivery system for mRNA vaccine after rational design and deserve further study on the in vivo delivery profile and anti-tumor efficacy.

Drug-drug conjugate nanoparticles (DDC NPs) is a potential method for overcoming poor solubility and nonspecific action in cancer therapy, which is based on its high drug loading efficiency and passive tumor-target properties. Our laboratory has prepared DOX-SS-DOX NPs based on disulfide-linked doxorubicin (DOX) drug-drug conjugate, which showed well physical stability and similar anti-tumor efficacy as liposomes. However, how structures of DDCs influence the self-assembling and anti-tumor efficacy is still seldom clarified and needs further investigation. Here, we discussed the role of linker types, length and linkage site in the NPs self-assembling and anti-tumor efficacy. A series of DOX prodrugs were prepared and all the prodrugs could self-assemble into NPs except DOX-SS-DOX (2), indicating the linker length played an important role during self-assembling process. The linkage sites and types of linker exhibited great influence on in vitro cytotoxicity and in vivo anti-tumor efficacy, particularly, modification on C-14 hydroxyl was more efficient for DOX release than on amino group. Besides, disulfide-bond was not cleaved and DOX-SH release did not occur in the metabolism process. The function of disulfide-bond was to enhance the release of DOX in the hydrolysis process. These findings is meaningful for effective prodrug NPs design for therapeutics. [ABSTRACT FROM AUTHOR]

Glucose isomerization to fructose is an important step in upgrading biomass to higher valued products. Tertiary amines can be highly selective catalysts for this reaction. In this work, tertiary amine analogues were functionalized on a mesoporous silica support to create heterogeneous catalytic materials. Experiments with homogeneous catalysts and silica revealed that silanols reduced the catalytic activity. Through using different strategies to limit amine-silanol interactions, it is determined that tuning parameters such as amine loading and alkyl linker length of the amine tether increases the fructose yield by a factor of four. Among the catalysts tested, the one found to be the best for glucose isomerization has a high amine loading (∼0.95 mmol g −1 ) with a methyl linker. Catalyst reuse tests demonstrated that organosilane leaching reduced amine content, but the immobilized amines with the methyl linker retained greater catalytic activity than amines with propyl linkers. [ABSTRACT FROM AUTHOR]

Peptide linkers of three different lengths were constructed to join the variable regions of the heavy chain (VH) and the light chain (VL) in a single-chain variable fragment antibody (scFv) specific for wogonin glucuronide (Wgn) that has the structure VH-(GGGGS) n –VL ( n = 3, 5, or 7). The scFv antibodies, which were expressed in Escherichia coli , were derived from an anti-Wgn monoclonal antibody (315A). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to evaluate their reactivity and sensitivity, which is also used for quantitative analysis of Wgn. Our results, showed that the reactivity and specificity of the three different scFvs were, in fact, similar. Subsequently, the scFv having a VH-(GGGGS) 3 –VL linker which was slightly better that other two scFvs against Wgn, was applied to indirect competitive ELISA (icELISA) to analyze Scutellariae Radix (S. Radix). The utility of the icELISA was demonstrated for quality control and analysis of S. Radix in this report. [ABSTRACT FROM AUTHOR]

The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL(n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using theBombyx morinucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs. Construction, expression, and characterization of multiform scFvs against daidzin (DZ-scFvs) with different linker lengths. [ABSTRACT FROM AUTHOR]

Nitro aromatics have broad applications in industry, agriculture, and pharmaceutics. However, their industrial production is faced with many challenges including poor selectivity, heavy pollution and safety concerns. Nature provides multiple strategies for aromatic nitration, which opens the door for the development of green and efficient biocatalysts. Our group's efforts focused on a unique bacterial cytochrome P450 TxtE that originates from the biosynthetic pathway of phytotoxin thaxtomins, which can install a nitro group at C4 of l-Trp indole ring. TxtE is a Class I P450 and its reaction relies on a pair of redox partners ferredoxin and ferredoxin reductase for essential electron transfer. To develop TxtE as an efficient nitration biocatalyst, we created artificial self-sufficient P450 chimeras by fusing TxtE with the reductase domain of the bacterial P450BM3 (BM3R). We evaluated the catalytic performance of the chimeras with different lengths of the linker connecting TxtE and BM3R domains and identified one with a 14-amino-acid linker (TB14) to give the best activity. In addition, we demonstrated the broad substrate scope of the engineered biocatalyst by screening diverse l-Trp analogs. In this chapter, we provide a detailed procedure for the development of aromatic nitration biocatalysts, including the construction of P450 fusion chimeras, biochemical characterization, determination of catalytic parameters, and testing of enzyme-substrate scope. These protocols can be followed to engineer other P450 enzymes and illustrate the processes of biocatalytic development for the synthesis of nitro chemicals., (Copyright © 2023. Published by Elsevier Inc.)

Highlights: [•] We investigated bispyridinium compounds in binding experiments with nAChRs. [•] We compared [3H]epibatidine affinity profiles to the inhibition of human AChE. [•] nAChR affinity and AChE inhibition depended on linker length. [•] Divalent cations enhanced [3H]epibatidine binding. [ABSTRACT FROM AUTHOR]

Highlights: [•] Controlling acid–base interactions is important in design of cooperative catalysts. [•] Homogeneous and heterogeneous results show weaker acids are beneficial. [•] Amine–silanol cooperativity tuned by adjusting the linker length and pore size. [•] Nitroaldol condensation is very sensitive to changes in amine–silanol cooperativity. [ABSTRACT FROM AUTHOR]

Abstract: α1-adrenoceptor (α1-AR) subtype-selective ligands lacking off-target affinity for the 5-HT1A receptor (5-HT1A-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2–12 atoms (C2–C12) for α1A, α1B and α1D -ARs and the 5-HT1A-R. These ligands are α1A-AR antagonists with nanomolar affinity for α1A and α1B -ARs. They display linker-length dependent selectivity for α1A/B -ARs over α1D-AR and the 5-HT1A-R. The C2 diquinoline has the highest affinity for α1A-AR (pK i 7.60±0.26) and greater than 30-fold and 600-fold selectivity for α1A-AR over α1D-AR and 5-HT1A-R respectively. A decrease in affinity for α1-ARs is observed as the linker length increases, reaching a nadir at 5 (α1A/1B-ARs) or 6 (α1D-AR) atoms; after which affinity increases as the linker is lengthened, peaking at 9 (α1A/1B/1D-ARs) or 8 (5-HT1A-R) atoms. Docking studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited α1D-AR and 5-HT1A-R affinity of these compounds makes them promising leads for future drug development of α1A-AR selective ligands without α1D-AR and the 5-HT1A-R off-target activity. [Copyright &y& Elsevier]

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N-terminus to N-terminus, C-terminus to C-terminus, and N-terminus to C-terminus (head-to-tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α-amino groups of the C-terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head-to-tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: Peptides involving the RGD motive (arginine–glycine–aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for αvβ3 integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG n ) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EG n linkers were investigated on their binding properties to cultured αvβ3 integrin-expressing U87MG cells. Methods: We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with 125I-labeled echistatin. Results: While c(RGDyK) function is a relative weak competitor against [125I]echistatin (K i, 329±18 nM) for αvβ3 integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K i, 64±23 nM). This effect was even more pronounced with the RGD trimers (K i, 40±7 nM) and tetramers (K i, 26±9 nM). The introduction of EG n spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [125I]echistatin. The EG 6 group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer). Conclusion: The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [125I]echistatin with the introduction of EG n spacers. This effect may be related to the decrease of the effective RGD molarity, which becomes most prominent within the tetravalent series. [Copyright &y& Elsevier]

Adopting carbon nanotube (CNT) transistors as biosensors has been developed as a promising method for cancer biomarker detection, which has shown superior sensitivity and selectivity. However, the detection of circulating tumor DNA (ctDNA) by the CNT transistor based biosensors is still a challenge and no work has been reported. Here, direct label-free DNA detection of AKT2 gene related to triple-negative breast cancer by all-CNT thin-film transistor (TFT) biosensors incorporated with tetrahedral DNA nanostructures (TDNs) is proposed and achieved for the first time. The adoption of TDNs enables improved biosensor response for at least 35% and even as high as 98% as compared with single-stranded DNA (ssDNA) probes owing to the enhanced DNA hybridization efficiency. Influence of the TDNs' linker length on the biosensor performance is important and has been investigated. Concentration-dependent DNA detection is achieved by the all-CNT TFT biosensors with a broad linear detection range of six orders of magnitude and a theoretical limit of detection (LOD) of 2 fM. In addition, the all-CNT TFT biosensors exhibit favorable selectivity and repeatability. The platform of all-CNT TFT biosensors incorporated with TDNs has great potential for multiplexed detection of various cancer biomarkers, providing a simple yet high performance universal strategy for low-cost clinical applications. • A tetrahedral DNA nanostructure (TDN) based all-carbon nanotube thin-film transistor biosensor was developed. • The adoption of TDNs enables improved biosensor response for as high as 98% as compared with single-stranded DNA probes. • DNA detection of AKT2 gene was achieved with a linear detection range from 1 pM to 1 μM and a detection limit of 2 fM. • The effect of TDNs' linker length on the biosensor performance was investigated. [ABSTRACT FROM AUTHOR]

Abstract: Development of tumor targeting pharmaceuticals on a modular platform is an attractive paradigm. Design choices for bispecific (anti-tumor and anti-chelate) pretargeting molecules are increased by the use of scFvs. Because a scFv is monovalent and small in size, its functional affinity and in vivo residence time can be improved through multimerization. ScFv multimers can be covalent or non-covalent. In vivo studies indicate that covalent scFv multimers are preferable. Attachment of scFv modules to scaffolds offers a wide range of possibilities for size and valency. A free thiol introduced at the C terminal end of a scFv (scFv-SH) allows for site-specific covalent attachment to a PEG scaffold without interfering with its antigen (Ag) binding. Although in theory, multimerization of 3 or 4 scFvs can be achieved by direct conjugation, as scFv-SH, to a tri or tetrafunctionalized PEG, it is not a practical option since homogeneous tri and tetrafunctionalized PEG are not readily available. However, the generation of (scFv)3–4-PEG molecules through attachment of combinations of di-scFv-SH (tandemly expressed scFvs) and scFv-SH or 2 di-scFv-SH to a bifunctional PEG is a sound approach that also allows for better control of the scFv-PEG conjugate molecular composition. Optimization of the molecular format of the di-scFv-SH module for production as soluble proteins in E. coli, Ag binding and conjugation is reported in this study. ScFvs in the VH-VL format were used for the di-scFv constructs since Fv domain inversion to VL-VH, while not yielding more protein, also abolished Ag binding. The effects on production yield, Ag binding and conjugation potential of the scFv joining linker length and the presence and location of an engineered cysteine were assessed in vitro. Our data indicate that for di-scFv-SH, an increase of the scFv joining linker length results in higher production and better Ag binding; a 20 aa long linker (G4S)4 was the longest linker tested. For the engineered cysteine, three locations were tested; within the scFv joining linker, at the C terminus upstream of the E Tag and as the carboxy terminal aa. The accessibility of the free SH assessed by conjugation of di-scFv-SH to HRP-Mal demonstrated that di-scFv-HRP conjugates are formed with comparable efficiencies when the cysteine is located at the scFv carboxy end. This empirical work provides a framework for the development of bispecific scFv multimers via site-specific attachment of scFv-SH and di-scFv-SH modules to a scaffold. [Copyright &y& Elsevier]

Our previous studies revealed that the nonapeptide fragment of HLA-DR molecule, located in the β chain 164–172 with the VPRSGEVYT sequence, suppresses the immune responses. The sequence is located on the exposed molecule loop, therefore it may be involved in the interactions with other proteins. We suggested that the loop may serve as a functional epitope on the HLA class II surface for intermolecular binding, and that possible mechanism of biological action of the synthesized peptides is associated with interfering of adhesion of HLA class II molecules to their coreceptors. It has been postulated that oligomerization of the coreceptors is required for stable binding to class II HLA. Based on the crystal dimeric structure of HLA-DR molecules, we designed, and synthesized molecules able to induce the putative coreceptors dimerization. The synthesized series of compounds consisted of two VPRSGEVYT sequences linked through their C-termini by spacers of different length: (VPRSGEVYTGn)2K-NH2 (n=4–6). The results demonstrate that the dimerization of the nonapeptide fragment of HLA-DR results in enhanced immunosuppressory properties. [Copyright &y& Elsevier]

[Display omitted] • The folding of a nucleosome array is a fundamental problem in chromatin biology. • The tri-nucleosome folding strongly depends on the linker length (LL). • The tri-nucleosome simulations with various LLs revealed 5 distinctive structures. • Those with LL ~ 10n takes a zigzag fiber form, while others do not. • The LL-dependence in the folding can solve a controversy in chromatin structure. The folding of a nucleosome array has long been one of the fundamental and unsolved problems in chromatin biology. In this study, we address how nucleosome array folding depends on the length of linker DNA. We performed molecular dynamics simulations of a tri-nucleosome, a minimal model of chromatin folding, with various linker lengths (LLs) ranging from 20 to 40 base pairs (bps). We found that the tri-nucleosome folding strongly depends on LLs, and classified the structure ensemble into five classes, named from trinuc-1 to trinuc-5. As a function of LL, the different classes appear, on average, every 2 bps with a period of 10 bps, and are characterized by distinct inter-nucleosome interactions. The trinuc-1 conformation corresponds to LL ~ 10n, where n is an integer, and is stabilized by the tight packing between the first and the third nucleosomes, consistent with a zigzag fiber form. Structures of the other four classes are more diverse and distributed continuously in the space of possible configurations. Histone-DNA electrostatic interactions in the tri-nucleosome are further analyzed. [ABSTRACT FROM AUTHOR]

• A streamlined, convenient, and modular two-enzyme complex was constructed. • The enzyme complex with the semirigid linker FRRRF showed the highest initial reaction rate. • Precise linker length and flexibility enable the efficient catalysis of the enzyme complex. Synthetic enzyme complexes have been successfully used to accelerate the reaction rate of cascade enzyme biocatalysis. Protein scaffold-mediated enzyme complexes are often constructed by assembling cascade enzymes on the artificial protein scaffoldin to form sophisticated biomimetic architectures and enhance the catalytic efficiency of biocatalytic processes. However, the effects of the linker in scaffoldin on the performance of the enzyme complexes have not been clarified. In this study, a scaffoldin-mediated two-enzyme complex containing triosephosphate isomerase (TIM) and fructose-1,6-bisphosphate aldolase/phosphatase (FBPA) was constructed, and the initial production rate of fructose 6-phosphate (F6P) was determined with different types of fine-tuning linkers. Enzyme complexes with linker length of 25 amino acids in scaffoldin exhibited the highest initial F6P production rate compared with linker length of 0, 10, or 57 amino acids in scaffoldin. This result indicated that an appropriate interdomain spacing between functional domains was required by multienzyme complexes to facilitate effective cascade catalysis. Then, the most popular flexible linker GGGGS (unit F) and rigid linker EAAAK (unit R) were introduced into this 25 amino acid linker to investigate the effect of linker flexibility on the initial reaction rate of the TIM–FBPA enzyme complex. The synthetic enzyme complex with the semirigid linker FRRRF in scaffoldin showed the highest initial F6P production rate of 10.16 μM/min, which indicates that the linker's amino acid composition in scaffoldin may lead to significant changes in the spatial architecture of the TIM–FBPA complex and consequently affect the initial reaction rate. Precise linker length and flexibility allow an appropriate interdomain conformation to enable efficient cascade reactions. Collectively, our results showed that fine-tuning the initial reaction rate of enzyme complexes is an integrated systematic engineering, including adjusting the multienzyme architecture, linker length, and linker flexibility, which provides rational guidance for designing effective multienzyme complexes in the future. [ABSTRACT FROM AUTHOR]

Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0-100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core-shell structured poly(lactic- co-glycolic acid)/poly(lactic acid)-PEG nanoparticles was evaluated using α v β 3 integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand-receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects.

Liposomes (LPs) as commonly used mRNA delivery systems remain to be rationally designed and optimized to ameliorate the antigen expression of mRNA vaccine in dendritic cells (DCs). In this study, we synthesized mannose-cholesterol conjugates (MP n -CHs) by click reaction using different PEG units (PEG 100 , PEG 1000 , and PEG 2000 ) as linker molecules. MP n -CHs were fully characterized and subsequently used to prepare DC-targeting liposomes (MP n -LPs) by a thin-film dispersion method. MP n -LPs loaded with mRNA (MP n -LPX) were finally prepared by a simple self-assembly method. MP n -LPX displayed bigger diameter (about 135 nm) and lower zeta potential (about 40 mV) compared to MP n -LPs. The in vitro transfection experiment on DC2.4 cells demonstrated that the PEG length of mannose derivatives had significant effect on the expression of GFP-encoding mRNA. MP 1000 -LPX containing MP 1000 -CH can achieve the highest transfection efficiency (52.09 ± 4.85%), which was significantly superior to the commercial transfection reagent Lipo 3K (11.47 ± 2.31%). The optimal DC-targeting MP 1000 -LPX showed an average size of 132.93 ± 4.93 nm and zeta potential of 37.93 ± 2.95 mV with nearly spherical shape. Moreover, MP 1000 -LPX can protect mRNA against degradation in serum with high efficacy. The uptake study indicated that MP 1000 -LPX enhanced mRNA expression mainly through the over-expressing mannose receptor (CD206) on the surface of DCs. In conclusion, mannose modified LPs might be a potential DC-targeting delivery system for mRNA vaccine after rational design and deserve further study on the in vivo delivery profile and anti-tumor efficacy.

Detailed data from statistical analyses of the structural properties of the inter-domain linker peptides of the bacterial regulators of the family MocR are herein reported. MocR regulators are a recently discovered subfamily of bacterial regulators possessing an N-terminal domain, 60 residue long on average, folded as the winged-helix-turn-helix architecture responsible for DNA recognition and binding, and a large C-terminal domain (350 residue on average) that belongs to the fold type-I pyridoxal 5'-phosphate (PLP) dependent enzymes such aspartate aminotransferase. Data show the distribution of several structural characteristics of the linkers taken from bacterial species from five different phyla, namely Actinobacteria, Alpha-, Beta-, Gammaproteobacteria and Firmicutes. Interpretation and discussion of reported data refer to the article "Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators" (T. Milano, S. Angelaccio, A. Tramonti, M. L. Di Salvo, R. Contestabile, S. Pascarella, 2016) [1].

Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro-Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β -turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties.

In a previous study, a novel cellobiohydrolase, PoCel6A, with new enzymatic activity against p-nitrophenyl-β-D-cellobioside (pNPC), was purified from Penicillium oxalicum. The cellulose-binding module and catalytic domain of PoCel6A showed a high degree of sequence similarity with other fungal Cel6As. However, PoCel6A had 11 more amino acids in the linker region than other Cel6As. To evaluate the relationship between the longer linker of PoCel6A and its enzymatic activity, 11 amino acids were deleted from the linker region of PoCel6A. The shortened PoCel6A linker nullified the enzymatic activity against pNPC but dramatically increased the enzyme's capacity for crystalline cellulose degradation. The shortened linker segment appeared to have no effect on the secondary structural conformation of PoCel6A. Another variant (PoCel6A-6pro) with six consecutive proline residues in the interdomain linker had a higher rigid linker, and no enzymatic activity was observed against soluble and insoluble substrate. The flexibility of the linker had an important function in the formation of active cellulase. The length and flexibility of the linker is clearly able to modify the function of PoCel6A and induce new characteristics of Cel6A., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The toxicity of organophosphorus nerve agents or pesticides arises from accumulation of acetylcholine and overstimulation of both muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) due to inhibition of acetylcholinesterase (AChE). Standard treatment by administration of atropine and oximes, e.g., obidoxime or pralidoxime, focuses on antagonism of mAChRs and reactivation of AChE, whereas nicotinic malfunction is not directly treated. An alternative approach would be to use nAChR active substances to counteract the effects of accumulated acetylcholine. Promising in vitro and in vivo results were obtained with the bispyridinium compounds SAD-128 (1,1'-oxydimethylene bis(4-tert-butylpyridinium) dichloride) and MB327 (1,1'-(propane-1,3-diyl)bis(4-tert-butylpyridinium) di(iodide)), which were partly attributed to their interaction with nAChRs. In this study, a homologous series of unsubstituted and 4-tert-butyl-substituted bispyridinium compounds with different alkane linker lengths was investigated in competition binding experiments using [(3)H]epibatidine as a reporter ligand. Additionally, the effect of the well-characterised MB327 on the [(3)H]epibatidine equilibrium dissociation (KD) constant in different buffers was determined. This study demonstrated that divalent cations increased the affinity of [(3)H]epibatidine. Since quaternary ammonium molecules are known to inhibit AChE, the obtained affinity constants of the tested bispyridinium compounds were compared with the inhibition of human AChE. In competition experiments, bispyridinium derivatives of longer linker length displaced [(3)H]epibatidine and inhibited AChE strongly. Bispyridinium compounds with short linkers, at most, have an allosteric interaction with the [(3)H]epibatidine binding sites and barely inhibited AChE. In dependence on alkane linker length, the bispyridinium compounds seemed to interact at different binding sites. However, the exact binding sites of the bispyridinium compounds responsible for the positive pharmacological effects have still not been identified, making predictive drug design difficult., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)