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Despite the widely reported success of consensus design in producing highly stabilized proteins, little is known about the physical mechanisms underlying this stabilization. Here we explore the potential sources of stabilization by performing a systematic analysis of the 29 substitutions that we previously found to collectively stabilize a consensus homeodomain compared to an extant homeodomain. By separately introducing groups of consensus substitutions that alter or preserve charge state, occur at varying degrees of residue burial, and occur at positions of varying degrees of conservation, we determine the extent to which these three features contribute to the consensus stability enhancement. Surprisingly, we find that the largest total contribution to stability comes from consensus substitutions on the protein surface and that the largest per-substitution contributions come from substitutions that maintain charge state. This finding suggests that although consensus proteins are often enriched in charged residues, consensus stabilization does not result primarily from interactions involving charged residues. Although consensus substitutions at strongly conserved positions also contribute disproportionately to stabilization, significant stabilization is also contributed from substitutions at weakly conserved positions. Furthermore, we find that identical consensus substitutions show larger stabilizing effects when introduced into the consensus background than when introduced into an extant homeodomain, indicating that synergistic, stabilizing interactions among the consensus residues contribute to consensus stability enhancement of the homeodomain. By measuring DNA binding affinity for the same set of variants, we find that although consensus design of the homeodomain increases both affinity and folding stability, it does so using a largely non-overlapping set of substitutions.

Centrin-1, a Ca2+ sensor protein of the centrin family is a crucial player for cell division in eukaryotes and plays a key role in the microtubule organising centre. Despite being regarded as a calcium sensor with a matched structure to calmodulin/troponin C, the protein undergoes mild changes in conformation and binds Ca2+ with moderate affinity. We present an in-depth analysis of the Ca2+ sensing by individual EF-hand motifs of centrin-1 and address unsolved questions of the rationales for moderate affinity and conformational transitions of the protein. Employing the more sensitive approach of Trp scanning of individual EF-hand motif, we have undertaken an exhaustive investigation of Ca2+ binding to individual EF-hand motifs, named EF1 to EF4. All four EF-hand motifs of centrin-1 are structural as all of them bind both Ca2+ and Mg2+. EF1 and EF4 are the most flexible sites as they undergo drastic conformational changes following Ca2+ binding, whereas EF3 responds to Ca2+ minimally. On the other hand, EF2 moves towards the protein surface upon binding Ca2+. The independent filling mode of Ca2+ to EF-hand motifs and lack of intermotif communication explain the lack of cooperativity of binding, thus constraining centrin-1 to a moderate affinity binding protein. Thus, centrin-1 is distinct from other calcium sensors such as calmodulin.

The most abundant member of the collagen protein family, collagen I (also known as type I collagen; COL1), is composed of one unique (chain B) and two similar (chain A) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contain a putative von Willebrand factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR, and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register.

Ochratoxin A (OTA) is a mycotoxin that can cause health risks to human/animal health. Contamination by OTA can occur in various foods and agricultural products, so sensitive and rapid detection of OTA is crucial. We describe a simple and sensitive fluorescence immunoassay for OTA using tetramethylrhodamine (TMR)-labeled OTA as a fluorescent probe. We conjugated tetramethylrhodamine to OTA through a covalent reaction, and obtained three TMR-OTA isomer probes after purification by high-performance liquid chromatography. All of the fluorescent probes showed high binding affinity to the anti-OTA antibody. Binding of the TMR-OTA probe to the antibody induced strong fluorescence of TMR-OTA due to the possible change in the local environment of TMR caused by affinity binding. In the presence of OTA, the OTA target competitively displaced the bound TMR-OTA probe from the antibody, causing a decrease in fluorescence. Measuring the change in fluorescence enabled rapid detection of OTA. This method was selective and allowed the detection of 1 nM OTA, showing potential for rapid OTA analysis in applications.

The effect of the ultrasonic treatment on the physicochemical properties, oil-holding capacities, foaming capacities, and TLR2-affinity of the polysaccharides from Pholiota nameko (PNPS), was evaluated. Compared with the protein content of PNPS before ultrasonic treatment, the protein content of PNPS all presented a decrease under different ultrasonic conditions. The viscosity of PNPS showed a decrease when the ultrasonic intensity was strong enough, as well as the molecular weight. The oil-holding capacity and the foaming capacity of PNPS showed a continuous increasing trend with the increase of the ultrasonic treatment time under a set ultrasonic power of 400 W. Further, the ultrasonic operation could induce the decrease of the affinity binding between PNPS and the receptor proteins TLR2. These data confirmed that applying the ultrasonic treatment could obtain PNPS with high carbohydrate contents, low viscosity and low TLR2-affinity under proper ultrasonic condition.

The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild-type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE-DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding.

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

Talin and vinculin are part of a multi-component system involved in mechanosensing in cell-matrix adhesions. Both exist in auto-inhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talin’s interaction with vinculin has not been investigated. Here by quantifying the force-dependent talin-vinculin interactions and kinetics using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an auto-inhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

Summary Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits.

Leishmaniasis considered as the most crucial epidemic-prone diseases according to the World Health Organization. Early diagnoses and therapy of Leishmania infection is a great challenge since, it has no symptom and is resistance to drugs. Therefore, there is an urgent need for sensitive and precise detection of this pathogen. In this study, a new method was developed for optical biosensing of Leishmania spp sequence based on hybridization of Citrate capped Ag nanoparticles bonded to specific single stranded DNA probe of Leishmania spp. Aggregation of the Citrate capped Ag nanoparticles in the existence or lack of a cDNA sequence of Leishmania, cause eye catching and considerable significant alter in the UV–vis. The obtained low limit of quantification (LLOQ) of was achieved as 1ZM. Based on experimental results in optimum conditions, quick bioanalysis of Leishmania spp sequence was performed (2 min). So, this probe can be used for the clinical diagnosis of this pathogen and infection disease., Analytical chemistry; Nanotechnology, Nanostructure; Affinity binding; Leishmaniasis; Spectrophotometer; Spectrofluorimetric; Biosensing

Amino acid substitutions at nonconserved protein positions can have non-canonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities (“DFI”) and dynamic coupling (“DCI”) for positions in the linker region of the lactose repressor protein (“LacI”). This region contains nonconserved positions for which substitutions alter DNA binding affinity. We first chose to study eleven substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA binding positions, and the degree of flexibility change correlated with experimentally-measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA binding positions in a manner that captured other experimentally-determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated almost no substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type LacI, the dynamic couplings between the DNA binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and non-canonical substitutions outcomes at nonconserved positions arise from re-wiring long-range communication among functionally-important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

Using frontal chromatography to determine the affinity binding constant (KPML) and the number of affinity binding sites (ΛPML) was demonstrated by the accuracy and precision affinity experiments of proteins (BSA, RNase, Cyt-C and Lys) on the iminodisuccinic acid metal chelate copper (IDS-Cu(II)-silica) column. The results displayed that R2 > 0.98 and RSD KPML(Cyt-C) > KPML(RNase) > KPML(BSA), and the values of KPML on the IDA-Cu(II)-silica column were larger than those on the IDS-Cu(II)-silica column, while the ΛPML values on the IDA-Cu(II)-silica column were smaller than those on the IDS-Cu(II)-silica column. The results of the KPML value were consistent with the affinity chromatographic behaviors of the proteins on the two columns, which proved the feasibility of the FC method again. Compared with ΛPML, the parameter KPML could better reflect the affinity strength of the proteins on the immobilized metal affinity chromatography (IMAC) column. The present work provided a fast and effective method for determining the binding parameters between proteins and IMAC columns, and also provided a theoretical basis for the separation and purification of proteins using the IMAC system.

In this review, we retrospect our progress in biological active naphthalimide and analogues as antitumor agents in the past 20 years. On one hand, various derivations in naphthalimide pharmacophores were developed to enhance their DNA binding affinity and antitumor property thereby. Heterocyclic fused naphthalimides, bis-naphthalimides, non-fused substituted naphthalimides and the carboxamide derivatives were synthesized. For example, thio-heterocyclic fused-naphthalimides were designed and evaluated in comparison with their oxo-heterocyclic fused analogues. Extended or created heterocycle-based skeleton were also developed as antitumor agents. On the other hand, we broaden the design strategy of naphthalimide antitumor agents besides DNA intercalation and topo II poison. We have introduced more drug design methods, such as prodrugs, multitarget drugs, computer-aided drug design, photodynamic therapy. For example, we have got naphthalimide derivatives which inhibited topo II and induced LMP by introducing long alkyl chain and polyamines. Several representative compounds were clarified of their antitumor mechanism of action. In all, our research improves the structure diversity of naphthalimide antitumor agents and distinct variances of antitumor targets and mechanism of action.

Astaxanthin (Ax), a type of carotenoid, has limited use as a result of its poor water solubility, low bioavailability, and decomposition under harsh conditions. This study reports a delivery system for Ax through a simple affinity binding with β-lactoglobulin and then coated with chitosan oligosaccharides. Ax-loaded β-lactoglobulin nanocomplexes and chitosan oligosaccharide-coated nanocomplexes were successfully prepared. The nanocomplexes exhibited a smooth spherical shape with diameters of about 40 and 60 nm measured by transmission electron microscopy. Spectroscopic techniques (ultraviolet-visible, fluorescence, and Fourier transform infrared spectroscopy) combined with molecular docking were used to determine the binding mechanism of Ax and β-lactoglobulin. In comparison to native Ax, the nanocomplexes maintain the hydroxyl radical scavenging activity of Ax under the treatment of acid, high temperature, and ultraviolet radiation. The release experiment of nanocomplexes revealed that the encapsulation could provide prolonged release of Ax in simulated gastrointestinal juices. This study aimed to fabricate and characterize Ax-β-lactoglobulin nanocomplexes, which can improve the Ax stability and slow release.

• An elastic degradable polyurethane (PU) bearing pendent HSNGLPL peptide for TGF-β1 affinity binding mimics the extracellular matrix function to retain and release growth factors.

Spatio-temporal presentation of growth factors is one of the key attributes of the cell's microenvironment. The design of macroporous alginate scaffolds, wherein TGF-b1 or BMP-4 is electrostatically bound to affinity binding sites of alginate sulfate, mimicking their presentation by the extracellular matrix (ECM), was previously shown to enable sustained presentation and release of each factor, thus increasing their biological activity. Specifically, TGF-b1/affinity-bound scaffolds induced the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded within these scaffolds. The prolonged activity of the affinity-bound TGF-b1 enabled efficient induction of signaling pathways leading to chondrogenesis, up to the appearance of committed chondrocytes. Similarly, BMP-4 affinity-bound to the macroporous alginate scaffold enabled efficient induction of osteogenic differentiation in hMSC constructs. Subsequent construction of a multicompartment inductive system, spatially-presenting TGF-b1 and BMP-4 in two distinct layers, enabled complete differentiation of hMSC to chondrocytes and osteoblasts, depending on the type of factor in use in the respective layer. This paper describes in detail the preparation method of the TGF-b1 or BMP4/affinity-bound alginate scaffolds, and the set of analyses performed to characterize the resultant scaffolds, including release profile study, released factor bioactivity, and functionality of the scaffolds as hMSC-inductive scaffolds.

Research has increasingly focused on the delivery of high, often excessive amounts of drugs, neglecting negative aspects of the carrier’s physical preconditions and biocompatibility. Among them, little attention has been paid to “small but beautiful” design of vehicle and multiple cargo to achieve effortless targeted delivery into deep tissue. The design of small biopolymers for deep tissue targeted delivery of multiple imaging agents and therapeutics (mini-nano carriers) emphasizes linear flexible polymer platforms with a hydrodynamic diameter of 4 nm to 10 nm, geometrically favoring dynamic juxtaposition of ligands to host receptors, and economic drug content. Platforms of biodegradable, non-toxic poly(β-l-malic acid) of this size carrying multiple chemically bound, optionally nature-derived or synthetic affinity peptides and drugs for a variety of purposes are described in this review with specific examples. The size, shape, and multiple attachments to membrane sites accelerate vascular escape and fast blood clearance, as well as the increase in medical treatment and contrasts for tissue imaging. High affinity antibodies routinely considered for targeting, such as the brain through the blood–brain barrier (BBB), are replaced by moderate affinity binding peptides (vectors), which penetrate at high influxes not achievable by antibodies.

The accurate assay of cardiac troponin I (cTnI) is very important for acute myocardial infarction (AMI), it also can be employed as an effective index for screening serious patients in COVID-19 pandemic before fatal heart injury to reduce the mortality. A ratiometric sensing strategy was proposed based on electrochemiluminescent (ECL) signal of doxorubicin (Dox)-luminol or the electrochemical (EC) signal of methylene blue (MB) vs. referable EC signal of Dox. The bio-recognitive Tro4-aptamer ensures the high specificity of the sensor by affinity binding to catch cTnI, and the tetrahedral DNA (TDs) on Au/Ti3C2-MXene built an excellent sensing matrix. An in situ hybrid chain reaction (HCR) amplification greatly improved the sensitivity. The ratiometric sensing responses ECLDox-luminol/CurrentDox or CurrentMB/CurrentDox linearly regressed to cTnI concentration in the range of 0.1 fM-1 pM or 0.1 fM-500 fM with the limit of detection (LOD) as 0.04 fM or 0.1 fM, respectively. Served as the reference signal, CurrentDox reflected the variation of sensor, it is very effective to ensure the accuracy of detection to obviate the false results. The proposed biosensors show good specificity, sensitivity, reproducibility and stability, have been applied to determine cTnI in real samples with satisfactory results. They are worth looking forward to be used for screening serious patient of COVID-19 to reduce the mortality, especially in mobile cabin hospital.

Fusion tags – amino acid sequences that are genetically coded to be expressed as attached moieties to a protein – have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the kcat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support.

The interaction of a novel macrocyclic copper(II) complex, ([CuL(ClO4)2] that L is 1,3,6,10,12,15-hexaazatricyclo[13.3.1.16,10]eicosane) with calf thymus DNA (ct-DNA) was investigated by various physicochemical techniques and molecular docking at simulated physiological conditions (pH = 7.4). The absorption spectra of the Cu(II) complex with ct-DNA showed a marked hyperchroism with 10 nm blue shift. The intrinsic binding constant (Kb) was determined as 1.25 × 104 M−1, which is more in keeping with the groove binding with DNA. Furthermore, competitive fluorimetric studies with Hoechst33258 have shown that Cu(II) complex exhibits the ability to displace the ct-DNA-bound Hoechst33258 indicating that it binds to ct-DNA in strong competition with Hoechst33258 for the groove binding. Also, no change in the relative viscosity of ct-DNA and fluorescence intensity of ct-DNA-MB complex in the present of Cu(II) complex is another evidence to groove binding. The thermodynamic parameters are calculated by van'...

Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability.

MXD proteins are transcription repressors that antagonize the E-box dependent activation of genes by MYC. MYC together with MIZ1 acts also as a repressor of a subset of genes, including cell cycle inhibitor genes such as p15 and p21. A role of MXDs in regulation of MYC-repressed genes is not known. Here we report that MXDs are functionally expressed in U2OS cells and activate transcription of p15 and p21, and other MYC-repressed genes. Activation of transcription was dependent on the interaction of MXDs with MIZ1, and on an intact DNA binding domain. MIZ1-binding deficient MXD mutants interacted with MAX and were active as repressors of MYC-activated genes but failed to activate MYC-repressed genes. Mutant MXDs with reduced DNA binding affinity interacted with MAX and MIZ1 but neither repressed nor activated transcription. Overexpression of MXDs attenuated proliferation of U2OS cells predominantly via MIZ1-dependent induction of p21. Our data show that MXDs and MYC have a reciprocally antagonistic potential to regulate transcription of mutual target genes.

Enzymes that generate ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products have garnered significant interest, given their ability to produce large libraries of chemically diverse scaffolds. Such RiPP biosynthetic enzymes are predicted to bind their corresponding peptide substrates through sequence-specific recognition of the leader sequence, which is removed after the installation of posttranslational modifications on the core sequence. The conservation of the leader sequence within a given RiPP class, in otherwise disparate precursor peptides, further supports the notion that strict sequence specificity is necessary for leader peptide engagement. Here, we demonstrate that leader binding by a biosynthetic enzyme in the lasso peptide class of RiPPs is directed by a minimal number of hydrophobic interactions. Biochemical and structural data illustrate how a single leader-binding domain can engage sequence-divergent leader peptides using a conserved motif that facilitates hydrophobic packing. The presence of this simple motif in noncognate peptides results in low micromolar affinity binding by binding domains from several different lasso biosynthetic systems. We also demonstrate that these observations likely extend to other RiPP biosynthetic classes. The portability of the binding motif opens avenues for the engineering of semisynthetic hybrid RiPP products.

We present an electrolyte-gated graphene field effect transistor (GFET) nanosensor using aptamer for rapid, highly sensitive and specific detection of a lung cancer biomarker interleukin-6 (IL-6) with enhanced stability. The negatively charged aptamer folds into a compact secondary conformation upon binding with IL-6, thus altering the carrier concentration of graphene and yielding a detectable change in the drain-source current Ids. Aptamer has smaller size than other receptors (e.g. antibodies), making it possible to bring the charged IL-6 more closely to the graphene surface upon affinity binding, thereby enhancing the sensitivity of the detection. Thanks to the higher stability of aptamer over antibodies, which degrade easily with increasing storage time, consistent sensing performance was obtained by our nanosensor over extended-time (>24 h) storage at 25 °C. Additionally, due to the GFET-enabled rapid transduction of the affinity recognition to IL-6, detection of IL-6 can be achieved in several minutes (

Stilbene derivatives have been found to possess promising anticancer activities against human cancer cell lines in vitro. In the present study, we have investigated cytotoxic, apoptosis induction and DNA binding activity of new stilbene derivative, (E)-1-(4-Chlorophenyl)-4,5-diphenyl-2-[4-(4-methoxystryl)phenyl]-1H-imidazol (STIM) on K562 chronic myeloid leukemia cell line. Via MTT assay STIM demonstrated cytotoxic activity against K562 cell line with IC

VP35 of Ebola viruses (EBOVs) is an attractive potential target because of its multifunction. All-atom molecular dynamics (MD) simulations and Molecular Mechanics Generalized Born surface area (MM/GBSA) energy calculations are performed to investigate the single-walled carbon nanotube (SWCNT) as an inhibitor in wild-type (WT) VP35 as well as in three primary mutants (K248A, I295A, and K248A/I295A) through docking the SWCNT in the first basic patch (FBP) of VP35. The SWCNTs of all the four systems effectively bind to the FBP. Interestingly, the sites and orientations of the SWCNT binding to the I295A mutant and K248A/I295A double mutants change significantly to accommodate the variation of the VP35 conformation. Moreover, the VDW can provide the major forces for affinity binding in all four systems.

Capacitive binding assays have long been considered highly sensitive. The underlying method in such assays includes sending short potential pulses and then registering the current. However, with the introduction of current pulses and registration of potential responses, the stability improved significantly, leading to assays of extreme sensitivities. The new assaying technology has been applied in a range of affinity binding assays, from immunoassays via DNA base pairing to the use of molecularly imprinted polymers (MIPs). Overall, very good sensitivities have been recorded, and the MIPs exhibit high stability and reusability.

5,10,15,20-tetra-(N-methyl-2-pyridyl)porphyrin (TMPyP2) was always paid little attention because it has relatively low DNA-binding affinity and photocytotoxicity compared with its positional isomer 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4). Inspired by the previous successful results on higher bioactivities of ligands with larger aromatic surfaces, herein, 5,10,15,20-tetra (phenyl-4-N-methyl-2-pyridyl) porphyrin (TPMPyP2), a TMPyP2-like porphyrin with enlarged planar substituents, was designed and successfully synthesized. Results from spectral experiments and molecular docking calculation suggest that TPMPyP2 could partially overcome the steric hindrance of TMPyP2, with more favorable DNA-binding mode and lower molecular binding energy (M.B.E). Meanwhile, cell photocytotoxicity research indicates that TPMPyP2 shows higher antitumor potency than TMPyP2, which allowed the previous conclusion that the higher G-Quadruplexes DNA binding affinity would be related to better photocytotoxicity.

In this paper, a colorimetric silver nanoparticles aptasensor (aptamer-AgNPs) was developed for simple and straightforward detection of protein in microfluidic chip. Surface-functionalized microfluidic channels were employed as the capture platform. Then the mixture of target protein and aptamer-AgNPs were injected into the microfluidic channels for colorimetric detection. To demonstrate the performance of this detection platform, thrombin was chosen as a model target protein. Introduction of thrombin could form a sandwich-type complex involving immobilized AgNPs. The amount of aptamer-AgNPs on the complex augmented along with the increase of the thrombin concentration causing different color change that can be analyzed both by naked eyes and a flatbed scanner. This method is featured with low sample consumption, simple processes of microfluidic platform and straightforward colorimetric detection with aptamer-AgNPs. Thrombin at concentrations as low as 20pM can be detected using this aptasensor without signal amplification. This work demonstrated that it had good selectivity over other proteins and it could be a useful strategy to detect other targets with two affinity binding sites for ligands as well.

We have reported earlier on the characterization of four peptides from a library of 15 peptide ligand candidates for human IgG (hIgG) obtained by a biomimetic design strategy and identified three high-affinity octapeptides. In this work, the left 11 peptides were evaluated and we found two more octapeptides (FYCHWQDE and FYCHNQDE) that showed high affinity for hIgG. The binding pH ranges for the two ligands were different, but the optimum pH values were the same for each other (pH 6.0). Both the ligands showed high specificity and bound hIgG mainly by electrostatic interactions. Ligand binding competition experiments revealed that the binding sites on hIgG for the two octapeptides were similar to those for Protein A. Finally, hIgG was purified from human serum with high purities and recovery yields with the two peptide affinity columns. Thus, among the 15 peptide candidates, a total of five octapeptides were identified as high-affinity ligands of hIgG. The five ligands were all derived from the same peptide model FYxHxxxE (where x denotes any amino acid) and contained four common hot spots F132, Y133, H137, and E143 of the affinity motif of Protein A. Analyses and evaluation of the peptide library would help deepen our understanding of the affinity binding of Protein A to IgG, and promote application of the biomimetic strategy in the design of affinity ligands for different proteins.

Molecular Mechanics-Generalized Born-Solvent Accessibility free energy calculations were used to analyse DNA binding affinity of 1-substituted carbazolyl-3,4-dihydro-β-carboline molecules. In this study, DNA structure with sequence of d(CGATCG)2 was used for simulations. 15 ns molecular dynamics simulations of the studied complexes were performed. The calculated free energy was compared with experimental antitumor activity (IC50). The predicted free energies decreased with the increase of IC50 values. It was shown that molecules 1–6 bind to DNA via intercalation mode, while molecules 7–9 bind through groove binding mode. Also, it was found that the vdW energy term (ΔE vdW) and the non-polar desolvation energy (ΔG SA) are the favorable terms for binding energy, whereas net electrostatic energies (ΔE ele + ΔG GB) and conformational entropy energy (TΔS) are unfavorable ones.

Prostate cancer is the most significant reason for deaths in men, outside of lung cancer. The clinical examination of cancer proteins or biomarkers is extremely significant in early examination and monitoring of recurrence of disease after treatment. Biomarkers have expanded great clinical significance owing to their extensive spectra in the identification, elimination, early diagnosis and cure of cancer. In this work, novel, ultrasensitive sandwich-type portable bio device based on citrate-capped silver nanoparticles (Citrate-AgNPs) modified graphene quantum dots (GQDs) nano ink for detection of Prostate specific antigen (PSA) was fabricated. Functionalized cysteamine with gold nanoparticles (Cys-AuNPs) was also utilized to amplify the signal. It provides a good and high external area for the immobilization biotinylated antibody of PSA in the large amount. For the first time, citrate-AgNPs-GQDs nano ink was directly written on the cellulose paper surface (ivory sheet and photographic paper) and modified by Cys-AuNPs. So, final structure of the immunodevices was completed after including of Ab1 and PSA (antigen). The immunosensors were used for the recognition of PSA by using DPVs (differential pulse voltammetry) technique. The obtained low limit of quantification (LLOQ) of the first immunodevice (ivory sheet/Citrate AgNPs-GQDs nano-ink/CysA-Au NPs/Ab1/BSA/PSA/Ab2) was 0.07 μg/L and the linear range for the calibration plot was from 0.07 to 60 μg/L. Also, the achieved LLOQ of the second immunodevice (photographic paper/Citrate AgNPs-GQDs nano-ink/Cys-Au NPs/Ab1/BSA/PSA/Ab2) was 0.05 μg/L with the linear range of 10 to 0.05 μg/L. It is noteworthy that, proposed immunoassay was effectively utilized to the monitoring of PSA glycoprotein in unprocessed human plasma sample. Obtained results show that the constructed immunosensor is able to apply as portable bio device for the clinical analysis of PSA in human plasma samples., Electrochemistry; Biomedical engineering; Cancer research; Immunology; Nanotechnology; Conductive ink; Affinity binding; Biomedical analysis; Immunodevice; Prostate cancer; Nanostructure

Summary Despite high sequence homology among the p53 family members, the regulation of their transactivation potential is based on strikingly different mechanisms. Previous studies revealed that the activity of TAp63α is regulated via an autoinhibitory mechanism that keeps inactive TAp63α in a dimeric conformation. While all p73 isoforms are constitutive tetramers, their basal activity is much lower compared with tetrameric TAp63. We show that the dimeric state of TAp63α not only reduces DNA binding affinity, but also suppresses interaction with the acetyltransferase p300. Exchange of the transactivation domains is sufficient to transfer the regulatory characteristics between p63 and p73. Structure determination of the transactivation domains of p63 and p73 in complex with the p300 Taz2 domain further revealed that, in contrast to p53 and p73, p63 has a single transactivation domain. Sequences essential for stabilizing the closed dimer of TAp63α have evolved into a second transactivation domain in p73 and p53.

A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO(2)) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.

MECP2 gene mutations can cause Rett Syndrome (RTT) – the second largest cause of mental retardation in girls. Studies based on the Methyl-Binding Domain (MBD) of MeCP2 and DNA complex crystals revealed how the mutated residues in the contact surface within DNA affect their binding. On the other hand, other mutations whose residue is not directly involved in the binding, are also related to RTT. In this paper, Molecular Dynamics (MD) and Potential of Mean Force (PMF) were applied to investigate how the MeCP2 P152R mutation influences MBD binding to DNA. The results suggest that the P152R mutation leads to MeCP2 increasing binding affinity towards both methylated and non-methylated DNA and decreasing binding selectivity towards methylated DNA. This may help explain previous inconclusive experimental results relating to the role of the P152R mutation in protein/DNA interactions and subsequent effects in RTT.

The binding constant is an important characteristic of a DNA-binding protein. A large number of methods exist to measure the binding constant, but many of those methods have intrinsic flaws that influence the outcome of the characterization. Tethered Particle Motion (TPM) is a simple, cheap, and high-throughput single-molecule method that can be used to reliably measure binding constants of proteins binding to DNA, provided that they distort DNA. In TPM, the motion of a bead tethered to a surface by DNA is tracked using light microscopy. A protein binding to the DNA will alter bead motion. This makes it possible to measure binding properties. We use the bacterial protein Integration Host Factor (IHF) as an example to show how specific binding to DNA can be measured. Moreover, we show a new intuitive quantitative approach to displaying data obtained via TPM.

It's favorable to alter KRas mutation's location to endomembrane by interfering the binding of PDEδ (the prenyl-binding protein phosphodiesterase delta) to KRas. In the present work, the binding of four inhibitors (Deltarasin, allyl analogue, pyrazolopyridazinone derivative, and Deltazinone 1) to PDEδ is investigated with all-atom Molecular Dynamic (MD) simulations. The binding free energy calculation results reveal that van der Waals (VDW) energy provides the major force for affinity binding. Moreover, the binding energy decomposition indicates that residues R61 and I129 provide important contributions to binding energies in all systems. The conserved hydrogen bonds play crucial roles in anchoring the inhibitors to the exact site for binding. The results for conformational analysis of PDEδ/free and PDEδ/inhibitors systems show that the structures are more stable after the inhibitors' binding to the PDEδ. It is also found that the most unstable system among four complexes is PDEδ/pyrazolopyridazinone derivative system whose α3-helix formed by the residues P113-Q116 disappears. This study may provide valuable information for the design of high potency PDEδ inhibitors. Communicated by Ramaswamy H. Sarma.

Laminin immobilization into diverse biological and synthetic matrices has been explored to replicate the microenvironment of stem cell niches and gain insight into the role of extracellular matrix (ECM) on stem cell behavior. However, the site-specific immobilization of this heterotrimeric glycoprotein and, consequently, control over its orientation and bioactivity has been a challenge that has limited many of the explored strategies to date. In this work, we established an affinity-based approach that takes advantage of the native high affinity interaction between laminin and the human N-terminal agrin (hNtA) domain. This interaction is expected to promote the site-selective immobilization of laminin to a specific substrate, while preserving the exposure of its key bioactive epitopes. Recombinant hNtA (rhNtA) domain was produced with high purity (>90%) and successfully conjugated at its N-terminal with a thiol-terminated poly(ethylene glycol) (PEG) without affecting its affinity to laminin. Self-assembled monolayers (SAMs) of mono-PEGylated rhNtA on gold (mPEG rhNtA-SAMs) were then prepared to evaluate the effectiveness of this strategy. The site-specific immobilization of laminin onto mPEG rhNtA-SAMs was shown to better preserve protein bioactivity in comparison to laminin immobilized on SAMs of thiol-PEG-succinimidyl glutaramide (HS-PEG-SGA), used for the non-selective covalent immobilization of laminin, as evidenced by its enhanced ability to efficiently self-polymerize and mediate cell adhesion and spreading of human neural stem cells. These results highlight the potential of this novel strategy to be used as an alternative to the conventional immobilization approaches in a wide range of applications, including engineered coatings for neuroelectrodes and cell culture, as well as biofunctionalization of 3D matrices. The mass spectrometry technique was performed at the Proteomics i3S Scientific Platform ( Roteiro/0028/2013 ; LISBOA-01-0145-FEDER-022125 ) with the assistance of Hugo Osório. IRRAS analysis was performed at the Biointerfaces and Nanotechnology Scientific Platform, with the assistance of Ricardo Vidal. In Cell Analyzer experiments were performed at the Biosciences Screening i3S Scientific Platform , member of the PPBI ( PPBI-POCI-01-0145-FEDER-022122 ) with the assistance of André Maia. Confocal microscopy was conducted at the Bioimaging i3S Scientific Platform , member of the PPBI ( PPBI-POCI-01-0145-FEDER-022122 ), with the assistance of Maria Lázaro. This work was supported by projects NORTE-01-0145-FEDER-000008 and NORTE-01-0145-FEDER-000012 , supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation ( POCI ), Portugal 2020; by Portuguese funds through FCT/MCTES in the framework of the project “Institute for Research and Innovation in Health Sciences” ( POCI-01-0145-FEDER-007274 ) and by project COMBINE (Santa Casa da Misericordia de Lisboa – Prémio Neurociências Mello e Castro). D.B. was supported by FCT PhD Programs ( PD/BD/105953/2014 ) and Programa Operacional Potencial Humano (POCH), in the scope of the BiotechHealth Program (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences), Programa FLAD Helthcare 2020 and the project PARES (Prémio Albino Aroso). Eduardo Conde-Sousa was supported by a post-doctoral grant of the project PPBI-POCI-01-0145-FEDER-022122 , in the scope of Fundação para a Ciência e Tecnologia, Portugal (FCT) National Roadmap of Research Infrastructures. Appendix A

Human noroviruses (HuNoVs) is the primary non-bacterial pathogen causing acute gastroenteritis worldwide. Molecular approaches have been mainly used for detection of HuNoVs. Aptamer-based assay has been also applied for detection of HuNoVs through affinity binding of viral capsid. In a conventional systematic evolution of ligands by exponential enrichment process, the target protein-bound sequences in the library were recovered by complicated process including affinity chromatography, extraction, membrane-filtration or antibody-conjugated magnetic beads. In this study, a novel approach was applied to select aptamers for HuNoVs. The new approach incorporated an in situ capture assay and next generation sequencing (NSG) for selecting the aptamers. P particles of HuNoV (GII.4) were purified and coated on the module to capture sequences that were specifically bound with the protein. The unbound sequences were easily removed by washing. The sequences with high affinity were amplified just in the wells and selected by repeated in situ selection process. From the total of 30,622,226 tested sequences, two aptamers, APTL-1 and APTL-6, were finally selected to incorporate with in situ capture RT-qPCR assay for detection of HuNoVs from clinical samples. The sensitivity of these two aptamers was compared with porcine gastric mucin (PGM) that contains well-known viral receptors, and the reported aptamer APT-M6-2. Both GI and GII HuNoVs could be detected from 5 clinical samples tested. The selected aptamer APTL-1 was comparable to PGM and slightly superior to the reported APTM6-2 aptamer for detection of HuNoVs from clinical samples. The results demonstrated that this in situ target-capture approach for aptamer selection is practicable.

The discovery of aptamers for aflatoxin B1 (AFB1), one of toxic carcinogens, has allowed to develop aptamer-based sensors and assays for aflatoxin. In this work, we reported a direct fluorescence anisotropy (FA) assay for investigation of aptamer-AFB1 binding and detection of AFB1 with the aptamer having single tetramethylrhodamine (TMR) label on a specific site. From a series of labeling sites of a 50-mer aptamer, we screened out the aptamer with TMR labeling at the 26th T, capable of generating good and large FA-decreasing response to AFB1. By using the T26-labeled 50-mer aptamer probe in FA analysis, we determined the affinity and selectivity of aptamer, and identified the crucial region of aptamer and optimum experimental conditions for strong binding. The aptamer could be further truncated to as short as 26 nucleotides in length, and this shorter aptamer possessed a simple stem-loop secondary structure and retained good binding affinity. Nucleotides in the loop region of the aptamer were conserved and important for affinity recognition. We achieved FA detection of AFB1 with a detection limit about 2 nM by using the TMR-labeled aptamer probe. The cross reactivity of aflatoxin B1, aflatoxin B2, aflatoxin M1, aflatoxin M2, aflatoxin G1, and aflatoxin G2 with aptamer were estimated to be 100%, 61%, 23%, 21%, 6.3%, 6.5%, respectively. The aptamer probe presented good selectivity over other mycotoxins and showed potential in complex sample analysis. This study of affinity binding between aptamer and aflatoxins will be helpful for developing other aptamer-based assays and sensors for aflatoxins.

Controlled delivery of heparin-binding (HB) proteins represents a challenge in regenerative medicine strategies. Here, we describe the features of novel nanoparticles (NPs), spontaneously coassembled due to affinity interactions between HB proteins and the semisynthetic anionic polysaccharide, alginate-sulfate. The NPs efficiently encapsulated and protected the proteins from proteolysis. Injection of a combination of NPs encapsulating multiple therapeutic growth factors promoted effective and long-term tissue repair in animal models of severe ischemia (murine model of hindlimb ischemia and acute myocardial infarction in rats). This simple yet efficient NP fabrication method is amenable for clinical use.

Surface functionalization of cellulosic materials using affinity binding macrobiomolecules is examined. Naturally evolved CBDIII from Clostridium thermocellum is showed efficient binding onto cotton fabrics, resulting in surfaces affording excellent water and contamination resistance. These results reveal a promising approach to fabricating biomaterials using interfacial assembling properties of biomolecules.

During the last two decades, cationic polymers have become one of the most promising synthetic vectors for gene transfection. However, the weak interactions formed between DNA and cationic polymers result in low transfection efficacy. Furthermore, the polyplexes formed between cationic polymers and DNA generally exhibit poor stability and toxicity because of the large excess of cationic polymer typically required for complete DNA condensation. Herein, we report the preparation of a novel class of bioreducible cationic nanomicelles by the use of disulfide bonds to connect the cationic shell to the fluorocarbon core. These bioreducible nanomicelles form strong interactions with DNA and completely condense DNA at an N/P ratio of 1. The resulting nanomicelle/DNA polyplexes exhibited high biocompatibility and performed very effectively as a gene-delivery system.

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Diamidine molecules, which have been recognized as the powerful gene drug candidates over the past decades, can bind in the DNA minor groove, inhibit the duplication of morbid sequences, and fight against a number of human and animal diseases. In this paper, on the basis of the binding models of a series of diamidines with DNA, the important influencing factors for the binding affinity of diamidines with DNA were systematically analyzed. The obtained results demonstrated that the curvature, length, distal group, and heteroaromatic ring of diamidine are four important factors, which could influence their binding affinities. Specifically, the better the curvature of the diamidine fits DNA minor groove, the higher the binding affinity is; increasing the molecular length within a certain range can make the binding affinity higher; changing the distal group of diamidine from amidino to imidazole or pyrimidine is favorable for improving the corresponding binding affinity; and the introduction of central heteroaromatic rings of diamidine molecules influences their binding affinities. One diamidine (named as DB103d) with ideal DNA binding affinity validates the four important factors proposed in the present work. The results obtained in this work might be helpful for the design of new efficient diamidine-based drug candidates.