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All biological processes rely on the formation of protein-ligand, protein-peptide and protein-protein complexes. Studying the affinity, kinetics and thermodynamics of binding between these pairs is critical for understanding basic cellular mechanisms. Many different technologies have been designed for probing interactions between biomolecules, each based on measuring different signals (fluorescence, heat, thermophoresis, scattering and interference, among others). Evaluation of the data from binding experiments and their fitting is an essential step towards the quantification of binding affinities. Here, user-friendly online tools to analyze biophysical data from steady-state fluorescence spectroscopy, microscale thermophoresis and differential scanning fluorimetry experiments are presented. The modules of the data-analysis platform (https://spc.embl-hamburg.de/) contain classical thermodynamic models and clear user guidelines for the determination of equilibrium dissociation constants (Kd) and thermal unfolding parameters such as melting temperatures (Tm).

Assuntos

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Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

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Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. An important class of ligands for the effective separation and purification of biotechnologically important substances is lectins, a group of naturally occurring molecules widely found in plants that display a range of specificities to bind different sugars. As sugars are often added to proteins through the process of glycosylation, ∼1/3 of all genetically encoded proteins are glycosylated, numerous cognate pairs of lectins with glycosylation groups have been discovered. Their specific binding interactions have not only allowed the development of numerous methodological strategies involving immobilized lectins to isolate molecules of interests but also for understanding the intermolecular interactions and alterations in glycosylation during a diverse set of biological phenomena, including tumor cell metastasis, intracellular communication, and inflammation. In this chapter, we describe a basic procedure for the separation of horse antibody classes by affinity chromatography based on differences in their glycosylation patterns. This procedure has been utilized for the purification of horse IgG3 (hoIgG3) from other six Ig from equine sera in a single step by using an Artocarpus integrifolia Jacalin column. This class of antibody comprises the therapeutic fraction generated in equine for passive antibody therapy and can serve as a biomarker for patient hypersensitivity. During the course of developing the protocol, the affinity interaction constant between the huIgE-hypersensitive immunoglobulin and the purified hoIgG3 was also determined.

Assuntos

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The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A5 (CA5) and A6 (CA6) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA5 or CA6. After elution, bound material was analyzed by UPLC-MS and CA5 was recovered from TBX2-loaded resins. To confirm and quantify the affinity (KD) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA5 and CA6 modified the thermophoretic behavior of TBX2, with a KD in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA5 was consistently more potent than CA6 in all tested cell lines with IC50 values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA5 by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA5 than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA5 may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.

RESUMO

Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (ß-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.

Assuntos

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Feline immunodeficiency virus (FIV) is a member of the retroviridae family of viruses. It causes acquired immunodeficiency syndrome (AIDS) in worldwide domestic and non-domestic cats and is a cause of an important veterinary issue. The genome organization of FIV and the clinical characteristics of the disease caused by FIV are similar to human immunodeficiency virus (HIV). Both viruses infect T lymphocytes, monocytes, and macrophages, with a similar replication cycle in infected cells. Thus, the infection of cats with FIV is also a useful tool for the study and development of novel drugs and vaccines against HIV. Anti-retroviral drugs studied extensively with regards to HIV infection have targeted different steps of the virus replication cycle: (1) disruption of the interaction with host cell surface receptors and co-receptors; (2) inhibition of fusion of the virus and cell membranes; (3) blocking of the reverse transcription of viral genomic RNA; (4) interruption of nuclear translocation and integration of viral DNA into host genomes; (5) prevention of viral transcript processing and nuclear export; and (6) inhibition of virion assembly and maturation. Despite the great success of anti-retroviral therapy in slowing HIV progression in humans, a similar therapy has not been thoroughly investigated for FIV infection in cats, mostly because of the little structural information available for FIV proteins. The FIV capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. During this step, the CA protein oligomerizes to form a protective coat that surrounds the viral genome. In this work, we perform a large-scale screening of four hundred molecules from our in-house library using an in vitro assembly assay of p24, combined with microscale thermophoresis, to estimate binding affinity. This screening led to the discovery of around four novel hits that inhibited capsid assembly in vitro. These may provide new antiviral drugs against FIV.

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Optical printing holds great potential to enable the use of the vast variety of colloidal nanoparticles (NPs) in nano- and microdevices and circuits. By means of optical forces, it enables the direct assembly of NPs, one by one, onto specific positions of solid surfaces with great flexibility of pattern design and no need of previous surface patterning. However, for unclear causes it was not possible to print identical NPs closer to each other than 300 nm. Here, we show that the repulsion restricting the optical printing of close by NPs arises from light absorption by the printed NPs and subsequent local heating. By optimizing heat dissipation, it is possible to reduce the minimum separation between NPs. Using a reduced graphene oxide layer on a sapphire substrate, we demonstrate for the first time the optical printing of Au-Au NP dimers. Modeling the experiments considering optical, thermophoretic, and thermo-osmotic forces we obtain a detailed understanding and a clear pathway for the optical printing fabrication of complex nano structures and circuits based on connected colloidal NPs.

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Optical printing is a simple and flexible method to bring colloidal nanoparticles from suspension to specific locations of a substrate. However, its application has been limited to the fabrication of arrays of isolated nanoparticles because, until now, it was never possible to bring nanoparticles closer together than approximately 300 nm. Here, we propose this limitation is due to thermophoretic repulsive forces generated by plasmonic heating of the NPs. We show how to overcome this obstacle and demonstrate the optical printing of connected nanoparticles with well-defined orientation. These experiments constitute a key step toward the fabrication by optical printing of functional nanostructures and microcircuits based on colloidal nanoparticles.

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Dihydroxynaphthyl aryl ketones 1-5 have been evaluated for their abilities to inhibit microtubule assembly and the binding to tubulin. Compounds 3, 4 and 5 displayed competitive inhibition against colchicine binding, and docking analysis showed that they bind to the tubulin colchicine-binding pocket inducing sheets instead of microtubules. Remarkable differences in biological activity observed among the assayed compounds seem to be related to the structure and position of the aryl substituent bonded to the carbonyl group. Compounds 2, 3 and 4, which contain a heterocyclic ring, presented higher affinity for tubulin compared to the carbocyclic analogue 5. Compound 4 showed the best affinity of the series, with an IC50 value of 2.1 µM for microtubule polymerization inhibition and a tubulin dissociation constant of 1.0 ± 0.2 µM, as determined by thermophoresis. Compound 4 was more efficacious in disrupting microtubule assembly in vitro than compound 5 although it contains the trimethoxyphenyl ring present in colchicine. Hydrogen bonds with Asn101 of α-tubulin seem to be responsible for the higher affinity of compound 4 respects to the others.

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Thermophoresis is an efficient process for the manipulation of molecules and nanoparticles due to the strong force it generates on the nanoscale. Thermophoresis is characterized by the Soret coefficient. Conventionally, the Soret coefficient of nanosized species is obtained by fitting the concentration profile under a temperature gradient at the steady state to a continuous phase model. However, when the number density of the target is ultralow and the dispersed species cannot be treated as a continuous phase, the bulk concentration fluctuates spatially, preventing extraction of temperature-gradient induced concentration profile. The present work demonstrates a strategy to tackle this problem by superimposing snapshots of nanoparticle distribution. The resulting image is suitable for the extraction of the Soret coefficient through the conventional data fitting method. The strategy is first tested through a discrete phase model that illustrates the spatial fluctuation of the nanoparticle concentration in a dilute suspension in response to the temperature gradient. By superimposing snapshots of the stochastic distribution, a thermophoretic depletion profile with low standard error is constructed, indicative of the Soret coefficient. Next, confocal analysis of nanoparticle distribution in response to a temperature gradient is performed using polystyrene nanobeads down to 1e-5% (v/v). The experimental results also reveal that superimposing enhances the accuracy of extracted Soret coefficient. The critical particle number density in the superimposed image for predicting the Soret coefficient is hypothesized to depend on the spatial resolution of the image. This study also demonstrates that the discrete phase model is an effective tool to study particle migration under thermophoresis in the liquid phase.