Use Of Differential Scanning Fluorimetry Research Articles

Characterization and optimization of fluorescein isothiocyanate labeling of humanized h2E2 anti-cocaine mAb

Fluorescein isothiocyanate (FITC) is widely used to fluorescently label reactive lysine residues on proteins, including antibodies. The rate and extent of labeling varies with reaction conditions, concentration of label, and the concentration and nature of the protein. Fluorescently labeled proteins are very useful, and one use for FITC labeled mAbs is development of assays to measure anti-mAb antibodies produced in vivo during treatment with antibody therapeutics. Our laboratory has developed a humanized anti-cocaine mAb (h2E2) intended for the treatment of cocaine use disorders. Thus, a well characterized FITC labeled h2E2 mAb is needed to quantitate possible anti-mAb antibodies. The time course of labeling and the relative incorporation of FITC into the heavy and light chains, as well as into the Fab and Fc portions of the mAb, was assessed. A novel use of differential scanning fluorimetry in the absence of any extrinsic fluorophore was developed and demonstrated to be capable of measuring antigen (cocaine) binding. In addition, the effect of increasing degrees of labeling by FITC on the thermodynamic parameters driving the binding of cocaine to the mAb was assessed via isothermal titration calorimetry (ITC). This binding technique, unlike others developed recently to measure cocaine binding, is not dependent on, or subject to interference by, the absorbance or fluorescence of the incorporated FITC label. The methods and results reported herein guide the optimization of FITC labeling needed for anti-mAb assays and other assays important for the development of therapeutic mAbs, which are some of the most specific and clinically useful drugs available.

The Application of Differential Scanning Fluorimetry in Exploring Bisubstrate Binding to Protein Arginine N ‐Methyltransferase 1

Protein arginine N-methyltransferases (PRMTs) are a family of 9 enzymes that catalyze mono- or di-methylation of arginine residues using S-adenosyl-l-methionine (SAM). Arginine methylation is an important post-translational modification that can regulate the activity and structure of target proteins. Altered PRMT activity can lead to a variety of health issues including neurodevelopmental disease, autoimmune disorders, cancer, and cardiovascular disease. Thus, developing a robust mechanistic understanding of PRMT function may provide insight into these various disease states and enable the development of potential therapeutic agents. Although PRMTs have been studied for nearly two decades, a consensus regarding the mechanism of action for this class of enzymes has remained noticeably elusive. To address this shortcoming, differential scanning fluorimetry (DSF) was used to gain mechanistic insight into the order of PRMT substrate and cofactor binding. This methodology confirms that PRMT cofactor binding precedes target substrate binding and supports the use of DSF to study bisubstrate enzymatic reaction mechanisms.

An AI-Powered Blood Test to Detect Cancer Using NanoDSF.

Simple SummaryBrain cancers, such as gliomas, are very difficult to detect because of their localization and late onset of symptoms. Here, we have developed a novel cancer detection method based on plasma denaturation profiles obtained by a non-conventional use of Differential Scanning Fluorimetry. Using blood samples from glioma patients and healthy controls, we show that their denaturation profiles can be automatically distinguished with the help of machine learning algorithms with 92% accuracy. This promising approach can now be extended to other types of cancers and could become a powerful pan-cancer diagnostic and monitoring tool requiring only a simple blood test.Glioblastoma is the most frequent and aggressive primary brain tumor. Its diagnosis is based on resection or biopsy that could be especially difficult and dangerous in the case of deep location or patient comorbidities. Monitoring disease evolution and progression also requires repeated biopsies that are often not feasible. Therefore, there is an urgent need to develop biomarkers to diagnose and follow glioblastoma evolution in a minimally invasive way. In the present study, we described a novel cancer detection method based on plasma denaturation profiles obtained by a non-conventional use of differential scanning fluorimetry. Using blood samples from 84 glioma patients and 63 healthy controls, we showed that their denaturation profiles can be automatically distinguished with the help of machine learning algorithms with 92% accuracy. Proposed high throughput workflow can be applied to any type of cancer and could become a powerful pan-cancer diagnostic and monitoring tool requiring only a simple blood test.

The Use of Differential Scanning Fluorimetry to Assess Strigolactone Receptor Function.

Differential scanning fluorimetry (DSF) is a method used for assessing the interaction of ligands with proteins. In most cases binding of a ligand to proteins tends to increase the melting temperature (Tm) of the protein involved. However, in the case of strigolactone receptors (e.g., D14, AtD14, DAD2, RMS3) from plants, the Tm tends to be reduced in the presence of strigolactones. This is likely due to increased flexibility of the receptors in the presence of hormone ligands.DSF experiments are simple, fast, amenable to high-throughput formats, and cost effective. They have therefore gained in popularity, including within the field of SL signaling. Typically in DSF the receptor protein is purified and incubated with the ligand (strigolactone, agonist, or antagonist) and a (fluorescent) reporter dye. The mixture is then placed in a quantitative PCR instrument and subjected to an increasing temperature gradient. Changes in fluorescence are recorded along the gradient, as the dye interacts with unfolded portions of the protein becoming accessible when the protein "melts". Differences in the temperature at which the protein unfolds in the absence and in the presence of the ligand are interpreted as indicating interactions between the ligand and the receptor.

Oligosaccharide Binding and Thermostability of Two Related AA9 Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharide substrates oxidatively. First discovered because of their action on recalcitrant crystalline substrates (chitin and cellulose), a number of LPMOs are now reported to act on soluble substrates, including oligosaccharides. However, crystallographic complexes with oligosaccharides have been reported for only a single LPMO so far, an enzyme from the basidiomycete fungus Lentinus similis (LsAA9_A). Here we present a more detailed comparative study of LsAA9_A and an LPMO from the ascomycete fungus Collariella virescens (CvAA9_A) with which it shares 41.5% sequence identity. LsAA9_A is considerably more thermostable than CvAA9_A, and the structural basis for the difference has been investigated. We have compared the patterns of oligosaccharide cleavage and the patterns of binding in several new crystal structures explaining the basis for the product preferences of the two enzymes. Obtaining structural information about complexes of LPMOs with carbohydrates has proven to be very difficult in general judging from the structures reported in the literature thus far, and this can be attributed only partly to the low affinity for small substrates. We have thus evaluated the use of differential scanning fluorimetry as a guide to obtaining complex structures. Furthermore, an analysis of crystal packing of LPMOs and glycoside hydrolases corroborates the hypothesis that active site occlusion is a very significant problem for LPMO-substrate interaction analysis by crystallography, due to their relatively flat and extended substrate binding sites.

Regulation of MALAT1 triple helix stability and in vitro degradation by diphenylfurans.

Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure–function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule–RNA targeting.

The application of differential scanning fluorimetry in exploring bisubstrate binding to protein arginine N-methyltransferase 1.

Protein arginine N-methyltransferases (PRMTs) are a family of 9 enzymes that catalyze mono- or di-methylation of arginine residues using S-adenosyl-l-methionine (SAM). Arginine methylation is an important post-translational modification that can regulate the activity and structure of target proteins. Altered PRMT activity can lead to a variety of health issues including neurodevelopmental disease, autoimmune disorders, cancer, and cardiovascular disease. Thus, developing a robust mechanistic understanding of PRMT function may provide insight into these various disease states and enable the development of potential therapeutic agents. Although PRMTs have been studied for nearly two decades, a consensus regarding the mechanism of action for this class of enzymes has remained noticeably elusive. To address this shortcoming, differential scanning fluorimetry (DSF) was used to gain mechanistic insight into the order of PRMT substrate and cofactor binding. This methodology confirms that PRMT cofactor binding precedes target substrate binding and supports the use of DSF to study bisubstrate enzymatic reaction mechanisms.

Common Challenges in Studying the Structure and Function of Bacterial Proteins: Case Studies from Helicobacter pylori.

Employing biophysical and structural methods is a powerful way to elucidate mechanisms of molecular recognition in bacterial pathogenesis. Such studies invariably depend on the production of pure, folded and stable proteins. Many proteins that can be expressed recombinantly ultimately fail to meet one or more of these criteria. The cag proteins from Helicobacter pylori form a secretion system that delivers the oncoprotein, CagA, into human gastric epithelial cells through an interaction between CagL and host cell integrins, where it can cause gastric adenocarcinoma. Expression of full length CagA and CagL is problematic as CagA undergoes rapid degradation during purification and CagL is thermally unstable. Here, we describe a method for the purification of CagA that results in the production of the full length protein through coexpression with its endogenous chaperone, CagF, and its subsequent separation from its chaperone. Furthermore, we detail the production of CagL and the use of differential scanning fluorimetry to identify how CagL is thermally stabilized by reduced pH, which led to a new crystal form of CagL and novel insight to pathogenic mechanisms. The methods described here for the production of stable cag proteins can be applied to a wide range of proteins involved in bacterial pathogenesis.

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry.

Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.

Use of Differential Scanning Fluorimetry to Identify Nuclear Receptor Ligands.

Identification of small molecules that interact specifically with the ligand-binding domains (LBDs) of nuclear receptors (NRs) can be accomplished using a variety of methodologies. Here, we describe the use of differential scanning fluorimetry to identify these ligands, a technique that requires no modification or derivatization of either the protein or the ligand, and uses an instrument that is becoming increasingly affordable and common in modern molecular biology laboratories, the quantitative, or real-time, PCR machine. Upon being introduced to specific ligands, nuclear receptors undergo structural and dynamic changes that tend to increase molecular stability, which can be measured by the resistance of the protein to heat denaturation. Differential scanning fluorimetry (DSF) uses a dielectric sensitive fluorescent dye to measure the thermal denaturation, or "melting" point (Tm) of a protein under different conditions, in this case in the absence and presence of a candidate ligand. Using DSF, multiple candidates can be screened at once, in numbers corresponding to plate size of the instrument used (e.g., 96- or 384-well), allowing significant throughput if a modest library of compounds needs to be tested.

The use of differential scanning fluorimetry in the rational design of plastic antibodies for protein targets.

The development of molecularly imprinted polymer nanoparticles (MIP-NPs), which specifically bind biomolecules, is of great interest in the area of biosensors, sample purification, therapeutic agents and biotechnology. Polymerisation techniques such as precipitation polymerisation, solid phase synthesis and core shell surface imprinting have allowed for significant improvements to be made in developing MIP-NPs which specifically recognise proteins. However, the development of MIP-NPs for protein templates (targets) still require lengthy optimisation and characterisation using different ratios of monomers in order to control their size, binding affinity and specificity. In this work we successfully demonstrated that differential scanning fluorimetry (DSF) can be used to rapidly determine the optimum imprinting conditions and monomer composition required for MIP-NP design and polymerisation. This is based on the stability of the protein template and shift in apparent melting points (Tm) upon interaction with different functional acrylic monomers. The method allows for the characterisation of molecularly imprinted nanoparticles (MIP-NPs) due to the observed differences in melting point profiles between, protein-MIP-NPs complexes, pre-polymerisation mixtures and non-imprinted nanoparticles (NIP-NPs) without the need for prior purification. The technique is simple, rapid and can be carried out on most quantitative polymerase chain reaction (qPCR) thermal cyclers which have the required filters for SYPRO

Use of differential scanning fluorimetry to optimize the purification and crystallization of PLP-dependent enzymes.

Differential scanning fluorimetry (DSF) is a practical and accessible technique that allows the assessment of multiphasic unfolding behavior resulting from subsaturating binding of ligands. Multiphasic unfolding is indicative of a heterogenous protein solution, which frequently interferes with crystallization and complicates functional characterization of proteins of interest. Along with UV-Vis spectroscopy, DSF was used to guide purification and crystallization improvements for the pyridoxal 5'-phosphate (PLP) dependent transaminase BioA from Mycobacterium tuberculosis. The incompatibility of the primary amine-containing buffer 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) and PLP was identified as a significant contributor to heterogeneity. It is likely that the utility of DSF for ligand-binding assessment is not limited to the cofactor PLP but will be applicable to a variety of ligand-dependent enzymes.

Use of Differential Scanning Fluorimetry as a High-Throughput Assay to Identify Nuclear Receptor Ligands

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10µL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.

Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10μL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.

Protein stability in an ionic liquid milieu: on the use of differential scanning fluorimetry

Protein stability is a major bottleneck in the biotechnological application of ionic liquid-containing solvents, either in the frame of biocatalysis or protein storage. Herein, differential scanning fluorimetry was successfully implemented as a high throughput method to fast scan the impact of a number of cholinium-based ionic liquids on the stability of proteins.

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.