Fluorescence-based Thermal Shift Assay Research Articles

From X-ray crystallographic structure to intrinsic thermodynamics of protein-ligand binding using carbonic anhydrase isozymes as a model system.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.

Inhibitor binding to metal-substituted metalloenzyme: Sulfonamide affinity for carbonic anhydrase IX

Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds.Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes.

Design, synthesis and biological evaluation of 2,4,6- trisubstituted triazine derivatives as new nonpeptide small-molecule SIRT5 inhibitors

Human sirtuin 5 (SIRT5) participates in a variety of metabolic disorder-associated diseases, including cancer. Inhibition of SIRT5 has been confirmed to provide a new strategy for treatment of related diseases. Previously, we discovered a pyrimidine skeleton inhibitor XIV, which showed low micromolar inhibitory activity against SIRT5. Herein, we utilized the scaffold-hopping strategy to design and synthesize a series of 2,4,6- trisubstituted triazine derivatives. The SAR analysis led to the discovery of several new SIRT5 inhibitors with low micromolar inhibition levels. The most potent compounds 10 (IC50 = 5.38 µM), and 14 (IC50 = 4.07 µM) were further confirmed to be the substrate-competitive SIRT5 inhibitors through enzyme kinetic assays, which is consistent with the molecular docking analyses. Fluorescence-based thermal shift assays proved that these compounds may stabilize SIRT5 by binding withprotein.. In addition, compounds 10 and 14 were also revealed to have moderate selectivity to SIRT5 over SIRT1-3. This study will aid further efforts to develop highly potent and selective SIRT5 inhibitors for the treatment of cancer and other related diseases.

Molecular and kinetic properties of copper nitrite reductase from Sinorhizobium meliloti 2011 upon substituting the interfacial histidine ligand coordinated to the type 2 copper active site for glycine

A copper-containing nitrite reductase catalyzes the reduction of nitrite to nitric oxide in the denitrifier Sinorhizobium meliloti 2011 (SmNirK), a microorganism used as bioinoculant in alfalfa seeds. Wild type SmNirK is a homotrimer that contains two copper centers per monomer, one of type 1 (T1) and other of type 2 (T2). T2 is at the interface of two monomers in a distorted square pyramidal coordination bonded to a water molecule and three histidine side chains, H171 and H136 from one monomer and H342 from the other. We report the molecular, catalytic, and spectroscopic properties of the SmNirK variant H342G, in which the interfacial H342 T2 ligand is substituted for glycine. The molecular properties of H342G are similar to those of wild type SmNirK. Fluorescence-based thermal shift assays and FTIR studies showed that the structural effect of the mutation is only marginal. However, the kinetic reaction with the physiological electron donor was significantly affected, which showed a ∼ 100-fold lower turnover number compared to the wild type enzyme. UV–Vis, EPR and FTIR studies complemented with computational calculations indicated that the drop in enzyme activity are mainly due to the void generated in the protein substrate channel by the point mutation. The main structural changes involve the filling of the void with water molecules, the direct coordination to T2 copper ion of the second sphere aspartic acid ligand, a key residue in catalysis and nitrite sensing in NirK, and to the loss of the 3 N-O coordination of T2.

Intrinsic affinity of protein – ligand binding by differential scanning calorimetry

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.

Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions.

Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.

Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability

A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein-ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.

Purification and characterization of an FeII- and α-ketoglutarate-dependent xanthine hydroxylase from Aspergillus oryzae

XanA is an FeII- and α-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 μM xanthine, 1 M NaCl, 600 μM α-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 °C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the N-terminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, α-ketoglutarate, and iron suggest that the enzyme is an iron- and α-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme's optimum activity conditions were determined to be 25 °C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

Inhibition of Tryptophan Hydroxylases and Monoamine Oxidase-A by the Proton Pump Inhibitor, Omeprazole-In Vitro and In Vivo Investigations.

Serotonin (5-HT) is a hormone and neurotransmitter that modulates neural activity as well as a wide range of other physiological processes including cardiovascular function, bowel motility, and platelet aggregation. 5-HT synthesis is catalyzed by tryptophan hydroxylase (TPH) which exists as two distinct isoforms; TPH1 and TPH2, which are responsible for peripheral and central 5-HT, respectively. Due to the implication of 5-HT in a number of pathologies, including depression, anxiety, autism, sexual dysfunction, irritable bowel syndrome, inflammatory bowel disease, and carcinoid syndrome, there has been a growing interest in finding modulators of these enzymes in recent years. We thus performed high-throughput screening (HTS) using a fluorescence-based thermal shift assay (DSF) to search the Prestwick Chemical Library containing 1,280 compounds, mostly FDA-approved drugs, for TPH1 binders. We here report the identification of omeprazole, a proton pump inhibitor, as an inhibitor of TPH1 and TPH2 with low micromolar potency and high selectivity over the other aromatic amino acid hydroxylases. The S-enantiomer of omeprazole, esomeprazole, has recently also been described as an inhibitor of monoamine oxidase-A (MAO-A), the main enzyme responsible for 5-HT degradation, albeit with lower potency compared to the effect on TPH1 and TPH2. In order to investigate the net effect of simultaneous inhibition of TPH and MAO-A in vivo, we administered high-dose (100 mg/kg) omeprazole to CD-1 mice for 4 days, after which the animals were subjected to the tail suspension test. Finally, central (whole brain) and peripheral (serum) 5-HT content was measured using liquid chromatography-mass spectrometry (LC-MS). Omeprazole treatment significantly increased 5-HT concentrations, both in brain and in serum, and reduced the time spent immobile in the tail suspension test relative to vehicle control. Thus, the MAO-A inhibition afforded by high-dose omeprazole appears to overcome the opposing effect on 5-HT produced by inhibition of TPH1 and TPH2. Further modification of proton pump inhibitor scaffolds may yield more selective modulators of 5-HT metabolism.

Expression, Purification, Refolding, and Characterization of a Neverland Protein From Caenorhabditis elegans.

Steroid hormones that serve as vital compounds are necessary for the development and metabolism of a variety of organisms. The neverland (NVD) family genes encode the conserved Rieske-type oxygenases, which are accountable for the dehydrogenation during the synthesis and regulation of steroid hormones. However, the His-tagged NVD protein from Caenorhabditis elegans expresses as inclusion bodies in Escherichia coli BL21 (DE3). This bottleneck can be solved through refolding by urea or the introduction of a maltose-binding protein (MBP) tag at the N-terminus. Through further research on purification after the introduction of a MBP tag at the N-terminus, the CD measurement and fluorescence-based thermal shift assay indicated that MBP was favorable for the NVD proteins’ solubility and stability, which may be beneficial for the large-scale manufacture of NVD protein for further research. The structural model contained the Rieske [2Fe–2S] domain and non-heme iron-binding motif, which were similar to 3-ketosteroid 9 α-hydroxylase.

The existence of a Cryptic Allosteric Site on Plasmodium falciparum Malate Dehydrogenase

Approximately one million people die from malaria each year, and thus this disease is a prevalent and pressing matter. Plasmodium falciparum, the protozoan parasite responsible for malaria has been subject to intensive study with the goal of developing specific drugs for malaria treatment. Plasmodium falciparum has a distinctive Malate Dehydrogenase (MDH), being tetrameric compared to the canonical dimeric forms of MDH, yet shares the same catalytic and substrate binding site motives as human mitochondrial and cytosolic forms. To investigate the possible existence of a cryptic allosteric site in the P falciparum enzyme, we have used sequence and structural bioinformatics and identified several short sequences,(G175‐L176, Q211‐M216) each containing highly conserved (in Plasmodium and related species) amino acids that are different in the Plasmodium falciparum enzyme than the human mitochondrial or cytosolic forms. To investigate the possibility that these regions form a cryptic allosteric site in Plasmodium falciparum we expressed, purified, using NiNTA Affinity chromatography, and characterized all three forms (Plasmodium falciparum, human cytosolic and human mitochondrial malate dehydrogenases). In terms of kinetic parameters the Plasmodium falciparum enzyme resembled the mammalian mitochondrial isoform, exhibiting similar substrate inhibition with oxaloacetate and weaker affinity for NADH than the mammalian cytosolic isoform. To determine the potential that the Plasmodium falciparum enzyme had a cryptic allosteric site we designed two mutations, one in each of the two significant sequence differences between the human isoforms and the Plasmodium falciparum enzyme (.D176N and R214E ). Each mutation was constructed using Quikchange mutagenesis, transformed into XL Gold cells and expressed. The purified mutant proteins were characterized using enzyme kinetics and size exclusion chromatography and their overall secondary structures compared to the wildtype enzyme using circular dichroism spectroscopy. Stability was compared using CD thermal melts at 222nm and a Fluorescence based Thermal Shift assay. While both mutations showed altered specific activities compared to wildtype, the D176N mutant showed a 90% lower activity than wildtype Plasmodium falciparum malate dehydrogenase. Both mutations significantly lowered affinity for the cofactor NADH, >20fold decrease in affinity. Taken together these results suggest that Plasmodium falciparum malate dehydrogenase may have a highly conserved cryptic allosteric site distinct from any such site in the mammalian isoforms. This Cryptic Allosteric Site could be exploited to develop inhibitors of Plasmodium falciparum malate dehydrogenase that would not impact the human isoforms.Support or Funding InformationThis work was supported by NSF Grants 1726932 and 0448905.

Small Molecule Inhibitors of the Response Regulator ArsR Exhibit Bactericidal Activity against Helicobacter pylori.

Helicobacter pylori is considered the most prevalent bacterial pathogen in humans. The increasing antibiotic resistance evolved by this microorganism has raised alarm bells worldwide due to the significant reduction in the eradication rates of traditional standard therapies. A major challenge in this antibiotic resistance crisis is the identification of novel microbial targets whose inhibitors can overcome the currently circulating resistome. In the present study, we have validated the use of the essential response regulator ArsR as a novel and promising therapeutic target against H. pylori infections. A high-throughput screening of a repurposing chemical library using a fluorescence-based thermal shift assay identified several ArsR binders. At least four of these low-molecular weight compounds noticeably inhibited the DNA binding activity of ArsR and showed bactericidal effects against antibiotic-resistant strains of H. pylori. Among the ArsR inhibitors, a human secondary bile acid, lithocholic acid, quickly destroyed H. pylori cells and exhibited partial synergistic action in combination with clarithromycin or levofloxacin, while the antimicrobial effect of this compound against representative members of the normal human microbiota such as Escherichia coli and Staphylococcus epidermidis appeared irrelevant. Our results enhance the battery of novel therapeutic tools against refractory infections caused by multidrug-resistant H. pylori strains.

Integrated Target-Based and Phenotypic Screening Approaches for the Identification of Anti-Tubercular Agents That Bind to the Mycobacterial Adenylating Enzyme MbtA.

Iron is essential for the pathogenicity and virulence of Mycobacterium tuberculosis, which synthesises salicyl-capped siderophores (mycobactins) to acquire this element from the host. MbtA is the adenylating enzyme that catalyses the initial reaction of mycobactin biosynthesis and is solely expressed by mycobacteria. A 3200-member library comprised of lead-like, structurally diverse compounds was screened against M. tuberculosis for whole-cell inhibitory activity. A set of 846 compounds that inhibited the tubercle bacilli growth were then tested for their ability to bind to MbtA using a fluorescence-based thermal shift assay and NMR-based Water-LOGSY and saturation transfer difference (STD) experiments. We identified an attractive hit molecule, 5-hydroxyindol-3-ethylamino-(2-nitro-4-trifluoromethyl)benzene (5), that bound with high affinity to MbtA and produced a MIC90 value of 13 μm. The ligand was docked into the MbtA crystal structure and displayed an excellent fit within the MbtA active pocket, adopting a binding mode different from that of the established MbtA inhibitor Sal-AMS.

Antibacterial activity of 2-amino-4-hydroxypyrimidine-5-carboxylates and binding to Burkholderia pseudomallei 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase.

Enzymes in the methylerythritol phosphate pathway make attractive targets for antibacterial activity due to their importance in isoprenoid biosynthesis and the absence of the pathway in mammals. The fifth enzyme in the pathway, 2-C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), contains a catalytically important zinc ion in the active site. A series of de novo designed compounds containing a zinc binding group was synthesized and evaluated for antibacterial activity and interaction with IspF from Burkholderia pseudomallei, the causative agent of Whitmore's disease. The series demonstrated antibacterial activity as well as protein stabilization in fluorescence-based thermal shift assays. Finally, the binding of one compound to Burkholderia pseudomallei IspF was evaluated through group epitope mapping by saturation transfer difference NMR.

A new herbicidal site of action: Cinmethylin binds to acyl-ACP thioesterase and inhibits plant fatty acid biosynthesis

The prevalent occurrence of herbicide resistant weeds increases the necessity for new site of action herbicides for effective control as well as to relax selection pressure on the known sites of action. As a consequence, interest increased in the unexploited molecule cinmethylin as a new solution for the control of weedy grasses in cereals. Therefore, the mechanism of action of cinmethylin was reevaluated. We applied the chemoproteomic approach cellular Target Profiling™ from Evotec to identify the cinmethylin target in Lemna paucicostata protein extracts. We found three potential targets belonging to the same protein family of fatty acid thioesterases (FAT) to bind to cinmethylin with high affinity. Binding of cinmethylin to FAT proteins from Lemna and Arabidopsis was confirmed by fluorescence-based thermal shift assay. The plastid localized enzyme FAT plays a crucial role in plant lipid biosynthesis, by mediating the release of fatty acids (FA) from its acyl carrier protein (ACP) which is necessary for FA export to the endoplasmic reticulum. GC–MS analysis of free FA composition in Lemna extracts revealed strong reduction of unsaturated C18 as well as saturated C14, and C16 FAs upon treatment with cinmethylin, indicating that FA release for subsequent lipid biosynthesis is the primary target of cinmethylin. Lipid biosynthesis is a prominent target of different herbicide classes. To assess whether FAT inhibition constitutes a new mechanism of action within this complex pathway, we compared physiological effects of cinmethylin to different ACCase and VLCFA synthesis inhibitors and identified characteristic differences in plant symptomology and free FA composition upon treatment with the three herbicide classes. Also, principal component analysis of total metabolic profiling of treated Lemna plants showed strong differences in overall metabolic changes after cinmethylin, ACCase or VLCFA inhibitor treatments. Our results identified and confirmed FAT as the cinmethylin target and validate FAT inhibition as a new site of action different from other lipid biosynthesis inhibitor classes.

Biophysical characterization of Suppressor of IKKepsilon structure, stability, and metal binding properties

Suppressor of IKKepsilon (SIKE) is phosphorylated in the signaling cascade that initiates the anti‐viral innate immune response mediated by Toll‐like receptor 3. Although shown to be associated with cytoskeletal proteins, the function of SIKE in association with the cytoskeleton or as part of the innate immune response is not yet known. To gain insight into SIKE's function, structural and biophysical characterization were undertaken. Fluorescence‐based thermal shift assays (FTSA) indicated that the SIKE structure displayed hydrophobic residues prior to thermal denaturation. With increasing temperature, additional hydrophobic character was displayed giving rise to unique FTS curves showing three transitions, initial binding of fluor, unfolding, and quenching of fluorescence, presumably by protein aggregation. From modeling software, a divalent cation binding site was predicted. Using FTSA, the addition of zinc and calcium appear to alter the thermal stability of SIKE. Using ANS as a reporter group or direct tryptophan fluorescence of SIKE, the binding affinity of SIKE to various divalent cations, zinc, magnesium, manganese and calcium, was assessed. Fluorescence quenching experiments with neutral and charged quenchers were used to probe the local environment of the lone Trp residue of SIKE. The quenching coefficient for the Trp in SIKE determined from acrylamide studies was significantly lower than that of free Trp in solution indicating that the residue is fairly protected. Together, these studies provide biophysical parameters with which to interrogate and refine our predicted SIKE structure that is being employed in interpreting how SIKE interacts with cytoskeletal proteins to mediate host defense mechanisms.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interactions between Divalent Cations and a Periplasmic Lipoprotein involved in Salmonella Magnesium Homeostasis

All living cells require magnesium to generate biochemical energy during metabolism and for maintenance of essential structures. A recently discovered protein of unknown function, DcrB, plays a role in magnesium homeostasis within Salmonella enterica, which must adapt to low external levels of Mg2+ to cause food‐borne illness. DcrB is a small protein attached to the periplasmic face of the cytoplasmic membrane. Given this location, one possible model for the function of DcrB is that DcrB brings Mg2+ to membrane Mg2+ transporters in the cytoplasmic membrane. Currently, Salmonella phenotypic assays have shown DcrB to promote growth in low Mg2+. The hypothesis of this project is that DcrB binds magnesium and possibly other divalent cations. To test this hypothesis, we examined whether Mg2+ and other divalent cations affected the thermal stability of purified DcrB protein. We examined the effect of divalent cations using a fluorescence‐based thermal shift assay and circular dichroism techniques. Furthermore, we are using our recent crystallographic structure of DcrB to identify and test specific metal binding sites. Our results have established the specificity of DcrB for binding divalent cations, which we anticipate will shed light on the role of DcrB in metal homeostasis in pathogenic bacteria.Support or Funding InformationThis research is supported by a University of Wisconsin‐‐La Crosse Undergraduate Research and Creativity Grant (RS) and a University of Wisconsin‐‐La Crosse Faculty Research Grant (JM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular and functional characterization of a glycosylated Galactose-Binding lectin from Mytilus californianus

Lectins play crucial roles for innate immune responses in invertebrates by recognizing and eliminating pathogens. In this study, a lectin from the mussel Mytilus californianus (MCL) was identified and characterized. The lectin was purified by affinity chromatography in α-lactose-agarose resin showing an experimental molecular mass of 18000 Da as determined by SDS-PAGE and MALDI-TOF mass spectrometry. It was specific for binding d-galactose and N-Acetyl-d-galactosamine that contained carbohydrate moieties that were also inhibited by melibiose and raffinose. It had the ability to agglutinate all types of human erythrocytes, as well as rabbit red blood cells. Circular dichroism analyzes have indicated that this lectin possessed an α/β fold with a predominance of β structures. This was consistent with the structure of the protein that was determined by the X-ray diffraction techniques. MCL was crystallized in the space group C21 and it diffracted to 1.79 Å resolution. Two monomers were found in the asymmetric unit and they formed dimers in solution. The protein has shown to be a member of the β-trefoil family, with three sugar binding sites per monomer. In accord with fluorescence-based thermal shift assays, we observed that the MCL Tm increased about 10 °C in the presence of galactose. Furthermore, we have determined the complete amino acid sequence by cDNA sequencing. The gene had two ORF2 proteins, one resulting in a 180 residue protein with a theoretical molecular mass of 20227 Da, and another resulting in a 150 residue protein with a theoretical molecular mass of 16911 Da. The difference between the theoretical and experimental values was due to the presence of a glycosylation that was observed by the glycosylation assay. A positive microbial agglutination and a growth inhibition activity were observed against Gram-negative and Gram-positive bacteria. The M. californianus lectin is the fourth member of the recently proposed new family of lectins that have been reported to date, occurring only in mollusks belonging to the family Mytilidae. It is the first member to be glycosylated and with a strong tendency to form large oligomers.

Characterization of Ligand Binding to Pseudokinases Using a Thermal Shift Assay.

The protocol herein describes a robust and proven method for the measurement of pseudokinase-ligand interaction using a fluorescence-based thermal shift assay (TSA). Pseudokinases are kinase-like proteins that have recently emerged as crucial regulatory modules of signal transduction pathways and may well represent a novel class of drug targets. However, unlike kinases, the regulatory activity of pseudokinases is mainly conferred through protein-protein interactions. Understanding the mechanisms that underlie pseudokinase conformational changes through ligand binding and how such conformational changes can tune signaling pathways is a necessary step to unravel their biological functions.Thermal denaturation-based methods have proven to be a powerful method for determining the capacity of purified recombinant pseudokinases to bind ligands and can simultaneously inform on the potential druggability of the nucleotide-binding site. This assay takes advantage of a change in fluorescence arising when the dye, SYPRO Orange, binds to hydrophobic patches that become exposed when a protein undergoes thermal unfolding. Ligand binding to a protein is known to increase its thermal stability, which is reflected by a shift between the thermal denaturation curves of the unliganded protein and the liganded protein. Here, we illustrate the utility of the method with the pseudokinases, ErbB3/HER3, ILK, ROP5Bi, JAK1, JAK2, TYK2, MLKL, STRAD, TRIB1, VRK3, and ROR1. This method can also be used to determine optimal buffer conditions that may increase protein stability and can be tailored to other protein families.

In Silico Discovery and Validation of Amide Based Small Molecule Targeting the Enzymatic Site of Shiga Toxin.

Shiga toxin (Stx), a category B biothreat agent, is a ribosome inactivating protein and toxic to human and animals. Here, we designed and synthesized small molecules that block the active site of the Stx A subunit. On the basis of binding energy, 20 molecules were selected for synthesis and evaluation. These molecules were primarily screened using fluorescence-based thermal shift assay and in vitro in Vero cells. Among 32 molecules (including 12 reported), six molecules offered protection with IC50 of 2.60-23.90 μM. 4-Nitro-N-[2-(2-phenylsulfanylethylamino)ethyl]benzamide hydrochloride is the most potent inhibitor with IC50 at 7.96 μM and selectivity index of 22.23 and is better than any known small molecule inhibitor of Stx. Preincubation with Stx offered full protection against Shiga toxin in mice. Surface plasmon resonance assay further confirmed that these molecules bind specifically to Stx A subunit. Further optimization is continued to identify a potential candidate which will be in vivo effective.