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Biotinylation of proteins, including antibodies, is a very useful and important modification for a variety of biochemical characterizations, including anti-drug antibody (ADA) assays used to detect antibodies raised against therapeutic antibodies. We assessed different degrees of biotin labeling of an anti-cocaine mAb currently under development for treating cocaine use disorder. We noted that higher levels of biotin labeling dramatically decreased mAb solubility, and increased the tendency to bind to surfaces, complicating characterization of the biotinylated antibody. Specifically, in phosphate buffered saline, labeling stoichiometries of more than about 3 biotin/mAb resulted in decreased recoveries due to increased binding to surfaces and decreased mAb solubility. Native gel agarose electrophoresis, differential scanning fluorimetry, and isothermal titration calorimetry all demonstrated changes in the mAb which became more pronounced above a labeling ratio of 3 biotin/mAb. At 3.0 biotin/mAb, there were minimal changes in solubility, adsorptivity, exposure of hydrophobic dye-binding sites, heat stability, and cocaine binding, in stark contrast to labeling with 5.6 biotin/mAb. Thus, the degree of biotinylation should be kept at about 3 biotin/mAb to maintain antigen binding and general structure, solubility, and stability of this mAb, a finding which may be important for other similar mAbs currently in use or under development. [Display omitted] • Anti-cocaine h2E2 mAb was biotinylated at levels from about 2 to 8 biotin/mAb. • Increasing biotinylation greatly reduced mAb solubility in PBS buffer. • At >3 biotin/mAb, reversible mAb adsorption caused variation in measured biotin/mAb. • DSF revealed large increases in hydrophobic mAb binding of Sypro dye at >3 biotin/mAb. • ITC revealed only small changes in mAb cocaine (antigen) binding at >3 biotin/mAb. [ABSTRACT FROM AUTHOR]

Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2. • Rab3A is preferentially phosphorylated by LRRK2 in its GDP-bound form. • Rab3A is resistant to heat denaturation at pH 7.4 and 9.0 but not at pH 5.0. • GTP-bound Rab3A is thermally stable compared with GDP-bound Rab3A. [ABSTRACT FROM AUTHOR]

[Display omitted] • TPPU_DSF easily calculates thermodynamic parameters from a single DSF measurement. • Compatible with common real time PCR instruments used for DSF measurements and high-throughput compound screening campaigns. • For the Web application no installation is required. It is compatible with all operating systems, it is free and open source and generates publication-ready figures and tables. • Data security. Data never leaves the user's computer, thus ensuring confidentiality. Here we present TPPU_DSF (https://maciasnmr.net/tppu%5fdsf/). This is a free and open-source web application that opens, converts, fits, and calculates the thermodynamic parameters of protein unfolding from standard differential scanning fluorimetry (DSF) data in an automated manner. The software has several applications. In the context of screening compound libraries for protein binders, obtaining thermodynamic parameters provides a more robust approach to detecting hits than the changes in the melting temperature (T m) alone, thereby helping to increase the number of positive hits in screening campaigns. Moreover, changes in ΔG u o indicate protein response to binding at lower compound concentrations than those in the T m , thereby reducing the costs associated with the amounts of protein and compounds required for the assays. Also, by adding thermodynamic information to the T m comparison, the software can contribute to the optimization of protein constructs and buffer conditions, a common practice before structural and functional projects. [ABSTRACT FROM AUTHOR]

Here we demonstrate that the antigen binding function of a humanized anti-cocaine mAb (h2E2) can be directly and easily determined using simple and inexpensive absorption spectroscopy and dyes commonly used for differential scanning fluorimetry, such as DASPMI and SYPRO Orange. Therapeutic monoclonal antibodies are commonly formulated in buffers which can interfere with necessary functional assays, containing additives and excipients such as mild detergents. Using the undiluted therapeutic product h2E2 mAb in its formulation buffer containing 0.01% polysorbate 80, the number of antigen/cocaine binding sites can be determined by the increase in absorbance (for DASPMI dye) or by the decrease in absorbance maximum wavelength (for SYPRO Orange dye), confirming proper function of the therapeutic mAb product. This ligand-induced visible dye absorption change can also be used to qualitatively evaluate the relative affinities of various metabolites of cocaine. These results are confirmed and extended by binding data obtained in the same formulation buffer using intrinsic tyrosine and tryptophan fluorescence quenching by cocaine, as well as by differential scanning fluorimetry. Interestingly, the binding of the cocaine metabolite norcocaine was demonstrated to be differentially affected by the pH 6 formulation buffer used for this mAb, presumably due to the differential ionizability of the demethylated norcocaine tropane ring nitrogen. This simple, direct, and inexpensive technique should prove useful for evaluation of other small molecule binding mAbs directly in their formulation buffers containing detergent, allowing rapid functional assessment of the produced therapeutic proteins. Image 1 • Cocaine binding of clinically formulated mAb is quantitated by visible dye absorption. • Functional ligand binding sites easily and rapidly measured by dye absorbance changes. • Cocaine metabolite relative affinities are qualitatively assessed using DASPMI dye. • This simple and direct method may be applicable to other small molecule directed mAbs. [ABSTRACT FROM AUTHOR]

• Differential scanning fluorimetry can be applied PRMT1 kinetic mechanism. • First-to-bind ligands stabilize the enzyme. • Second-to-bind ligands do not stabilize the enzyme. • Binding of two substrates contributes to an additive stabilizing effect. 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. [ABSTRACT FROM AUTHOR]

The β-galactoside-binding protein, galectin-3, is extensively involved in cancer development, progression and metastasis through multiple mechanisms. Inhibition of the galectin-3-mediated actions is increasingly considered as a promising therapeutic approach for cancer treatment. Our early studies have identified several novel galectin-3 binding inhibitors from chemical modification of the anticoagulant drug heparin. These heparin-derived galectin-3 binding inhibitors, which show no anticoagulant activity and bind to the galectin-3 canonical carbohydrate-binding site, induce galectin-3 conformational changes and inhibit galectin-3-mediated cancer cell adhesion, invasion and angiogenesis in vitro and reduce metastasis in mice. In this study, we determined the binding affinities of these heparin-derived ligands to galectin-3 using an isothermal titration calorimetry (ITC) ligand displacement approach. Such ITC experiments showed that the 2-de-O-sulphated, N-acetylated (compound E) and 6-de-O-sulphated, N-acetylated (F) heparin-derived ligands and their ultra-low molecular weight sub-fractions (E3 and F3) bind to galectin-3 with K D ranging from 0.96 to 1.32 mM.Differential scanning fluorimetry analysis revealed that, in contrast to the disaccharide ligand, N-acetyl-lactosamine, which binds to the fully folded form of galectin-3 and promotes galectin-3 thermal stability, the heparin-derived ligands preferentially bind to the unfolded state of galectin-3 and cause destabilization of the galectin-3 protein structure. These results provide molecular insights into the interaction of galectin-3 with the heparin-derived ligands and explain the previously demonstrated in vitro and in vivo effects of these binding inhibitors on galectin-3-mediated cancer cell behaviours. • The heparin derived inhibitors bind to galectin-3 with affinity at low mM range. • These inhibitors preferentially bind to the unfolded state of galectin-3. • Their binding de-stabilizes the galectin-3 protein structure. • A molecular insight of galectin-3 interaction with these novel binding inhibitors. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies are utilized for treating many diseases and disorders, as well as for basic research and development. Covalent labeling of mAbs is important for various antibody applications and creating antibody drug conjugates. Labeling at reactive lysine residues using lysine selective reagents is useful, but is non-selective and can interfere with antigen binding and interactions of the Fc antibody region. In this work, using an anti-cocaine mAb (h2E2), we utilized triphenylphosphine-3,3′,3″-trisulfonic acid (TPPTS), and demonstrated for the first time reduction of disulfides in an antibody by TPPTS. More importantly, this reduction was very reproducible, limited, and selective, and permitted selective labeling of the antibody with a cysteine reactive fluorescent reagent, resulting in labeling of a few specific cysteines. Similar results were obtained using TCEP-agarose reduction. We demonstrated that both of these selective partial reduction methods gave rise to approximately two labels per mAb, mostly by selective reduction of the heavy chain to light chain disulfide bond, as demonstrated by non-reducing SDS-PAGE protein band analysis. Thus, convenient, reproducible, and selective mAb disulfide reduction was achieved under mild conditions. These labeled, partially reduced mAbs were characterized by differential scanning fluorimetry (DSF), detecting the incorporated fluorescein instead of an exogenously added dye, and for antigen (cocaine) binding by isothermal titration calorimetry (ITC). Both the structure and antigen binding of the mAb was maintained. This novel selective reduction and labeling is generally relevant to modification of antibodies and to future development of conjugated mAbs for experimental and therapeutic purposes. [Display omitted] • Triphenylphosphine (TPPTS) was shown for the first time to reduce mAb disulfides. • Anti-cocaine h2E2 mAb was selectively partially reduced by TCEP-agarose or TPPTS. • Partially reduced mAb was labeled by 2 mol label/mol mAb via cysteine modification. • Cocaine binding to mAb assessed by differential scanning fluorimetry (DSF) and ITC. • Selective reduction and labeling had no negative effects on antigen (cocaine) binding. [ABSTRACT FROM AUTHOR]

The deviant Ras activation machinery is found in approximately 30% of all human cancers. SOS1 is an important protagonist of this pathway that plays a key-role in aberrant cell proliferation and differentiation. Interaction of SOS1 with 14-3-3 proteins modulates SOS1 activity in Ras-MAPK signaling. In the present study, we analyze the 14-3-3/SOS1 protein-protein interaction (PPI) by different biochemical assays and report the high resolution crystal structure of a 13-mer motif of SOS1 bound to 14-3-3ζ. These structural and functional insights are important for the evaluation of this PPI interface for small-molecule stabilization as a new starting point for modulating the Ras-Raf-MAPK pathway. [ABSTRACT FROM AUTHOR]

Src-homology-2-domain-containing protein tyrosine phosphatase-2 (SHP2) is a signaling enzyme whose activity is governed by an equilibrium between autoinhibited and activated states. Regulation of SHP2 activity is critical for cellular homeostasis, and mutations that alter its autoregulatory equilibrium cause cancers and developmental disorders. Several methods for assessing the strength of autoinhibitory interactions in SHP2 mutants have been previously reported, but each has limitations. We show that differential scanning fluorimetry provides a rapid, quantitative measure of SHP2 autoinhibition that is independent of the intrinsic activity of the SHP2 mutant being analyzed, does not involve protein labeling, and does not require specialized instrumentation. [Display omitted] • SHP2's activity is controlled by an interdomain autoinhibitory interaction. • Mutations that weaken SHP2's autoinhibition are causative for human pathologies. • Assessing SHP2 autoinhibition generally requires specialized equipment. • Differential scanning fluorimetry provides a rapid measure of SHP2 autoinhibition. • DSF does not require protein labeling or specialized instrumentation. [ABSTRACT FROM AUTHOR]

The use of enzymes in organic solvents has considerably widened their repertoire of applications. Such low water containing media also offer the possibility of carrying out enzymatic reactions at higher temperatures and enhancing reaction yields. The utility of such preparations is limited by the damage caused to the protein structure during freeze-drying. This work investigates the result of exposing the proteolytic enzyme subtilisin to high temperature in low water containing n-octane on its activity in aqueous and non-aqueous media. Exposing subtilisin at 90 °C for 5 h led to 18-fold improvement in its transesterification activity even at the normal assay temperature (37 °C) when compared with the untreated enzyme. The use of n-octane as the reaction medium was important as it helped to retain the three-dimensional architecture of the enzyme and should be considered while designing strategies for obtaining high activity preparations of other enzymes. Structural analysis using differential scanning fluorimetry showed that the enzyme lost its structure after heating in aqueous medium but retained it when heated in organic solvent. The simplicity and general applicability of the strategy should make it useful for obtaining highly active preparations of other enzymes as well. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Saw1 is an essential gene in single-strand annealing – the DNA repair pathway that repairs double-strand breaks when they occur between homologous repeats. Saw1 interacts with the structure-specific nuclease Rad1-Rad10 and this results in the recruitment of this nuclease to 3′ non-homologous DNA tailed recombination intermediates. Saw1 is unstable in the absence of the Rad1-Rad10 nuclease and, hence, it has been difficult to study its specific function in vitro. In the present work, we present the combination of dynamic light scattering and differential scanning fluorimetry techniques to optimize the stability and homogeneity of recombinant Saw1. The protein expression and purification conditions identified in this study allow for higher recovery of soluble Saw1 and enable the biochemical characterization of the protein. [ABSTRACT FROM AUTHOR]

The K i of carbonic anhydrase (CA) inhibitors is often determined by the stopped- flow CO 2 hydration assay, the method that directly follows the inhibition of CA enzymatic activity. However, the assay has limitations, such as largely unknown concentration of CO 2 and the inability to determine the K i below several nM. The widely used direct binding assay, isothermal titration calorimetry, also does not determine the K d below several nM. In contrast, the thermal shift assay can accurately determine picomolar affinities. New equations estimating CO 2 concentration were developed for the determination of k cat and K M of CA I and CA II. The inhibitor dose-response curves were analyzed using Hill and Morrison equations demonstrating that only the Morrison model is applicable for the determination of tight-binding inhibitor K i . The measurements of interactions between ten inhibitors and seven CA isoforms showed the limitations and advantages of all three techniques. Inhibitor 6 exhibited the K d of 50 pM and was highly selective towards human CA IX, an isoform which is nearly absent in healthy human, but highly overexpressed in numerous cancers. Combination of inhibition and binding techniques was necessary for precise determination of CA-high-affinity inhibitor interactions and future drug design. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding ( K nd ), and protein unfolding by urea ( I 50 ). This demonstrates that DSF methods can determine the structural stability of an enzyme's active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity. [ABSTRACT FROM AUTHOR]

Many bacterial respiratory redox enzymes depend on the twin-arginine translocase (Tat) system for translocation and membrane insertion. Tat substrates contain an N-terminal twin-arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone - redox enzyme maturation protein (REMP) - for final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin-arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase. In this study, we have utilized the in vitro proteineprotein interaction technique of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF) to investigate the interaction of guanosine nucleotides (GNPs) with DmsD. Here we have shown highly cooperative binding of DmsD with GTP. A dissociative ligandbinding style isotherm was generated upon GTP titration into the DmsD:DmsAL interaction, yielding sigmoidal release of DmsD with a Hill coefficient of 2.09 and a dissociation constant of 0.99 mM. DSF further illustrated the change in thermal stability upon DmsD interaction with DmsAL and GTP. These results imply the possibility of DmsD detection and binding of GTP during the DMSO protein maturation mechanism, from ribosomal translation to membrane targeting and final assembly. Conceivably, GTP is shown to act as a molecular regulator in the biochemical pathway. [ABSTRACT FROM AUTHOR]

InhA is an enoyl-ACP reductase of Mycobacterium tuberculosis implicated in the biosynthesis of mycolic acids, essential constituents of the mycobacterial cell wall. To date, this enzyme is considered as a promising target for the discovery of novel antitubercular drugs. In this work, we describe the first crystal structure of the apo form of the wild-type InhA at 1.80 Å resolution as well as the crystal structure of InhA in complex with the synthetic metabolite of the antitubercular drug isoniazid refined to 1.40 Å. This metabolite, synthesized in the absence of InhA, is able to displace and replace the cofactor NADH in the enzyme active site. This work provides a unique opportunity to enlighten the structural adaptation of apo-InhA to the binding of the NADH cofactor or of the isoniazid adduct. In addition, a differential scanning fluorimetry study of InhA, in the apo-form as well as in the presence of NAD + , NADH and INH-NADH was performed showing that binding of the INH-NADH adduct had a strong stabilizing effect. [ABSTRACT FROM AUTHOR]

Identification of the drug target of lead compounds is an important means for rapid and efficient drug discovery. Protein chips are a high-throughput protein function analysis technology that has been widely used in screening drug protein targets in recent years. However, the verification of the results after high-throughput protein chip screening is still cumbersome. Based on our mature protein chip preparation platform, we prepared a protein chip containing 150 important high-frequency protein targets and used antibodies to prove the availability of the protein chip. To improve the accuracy of target screening, we combined the label-free differential scanning fluorimetry (DSF) with the protein chip, proposing the Chip–DSF strategy. Subsequently, we tested the method with small molecular ginsenoside-Rg2 (Rg2). The Chip–DSF strategy was used to successfully screen the potential target protein KRAS(G12C) of Rg2. Consistently, we found that Rg2 could inhibit NCI-H23 cell proliferation by inducing cell cycle arrest. Also, we found that Rg2 could reduce the amount of KRAS protein and inhibit the phosphorylation of KRAS downstream key signaling protein ERK1, RPS6, and P70S6K in NCI-H23 cells. Collectively, our Chip-DSF strategy could achieve rapid target verification which improved the accuracy and efficiency of target screening of protein chips. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • A high-throughput focused DSF data analyzer, which runs very fast and in a highly efficient manner. • The software integrates plate-screening with protein–ligand affinity calculations. • Provides a Rank of all binders ordered according to differences in melting temperatures. • Generates Excel files and final K D reports automatically. The identification of new drugs for novel therapeutic targets requires the screening of libraries containing tens of thousands of compounds. While experimental screenings are assisted by high-throughput technologies, in target-based biophysical assays, such as differential scanning fluorimetry (DSF), the analysis steps must be calculated manually, often combining several software packages. To simplify the determination of the melting temperature (T m) of the target and the change induced by ligand binding (Δ T m), we developed the HTSDSF explorer, a versatile, all-in-one, user-friendly application suite. Implemented as a server-client application, in the primary screenings, HTSDSF explorer pre-analyzes and displays the T m and Δ T m results interactively, thereby allowing the user to study hundreds of conditions and select the primary hits in minutes. This application also allows the determination of preliminary binding constants (K D) through a series of subsequent dose–response assays on the primary hits, thereby facilitating the ranking of validated hits and the advance of drug discovery efforts. [ABSTRACT FROM AUTHOR]

Abstract: A simple, inexpensive, and universal method to quantify the recombinant proteins in Escherichia coli cell lysate using differential scanning fluorimetry (DSF) is reported. This method is based on the precise correlation between Δ(fluorescence intensity) determined by DSF and the amount of protein in solution. We first demonstrated the effectiveness of the DSF method using two commercially available enzymes, α-amylase and cellobiase, and then confirmed its utility with two recombinant proteins, amylosucrase and maltogenic amylase, expressed in E. coli. The Δ(fluorescence intensity) in DSF analysis accurately correlated with the concentration of the purified enzymes as well as the recombinant proteins in E. coli cell lysates. The main advantage of this method over other techniques such as Western blotting, enzyme-linked immunosorbent assay (ELISA), and green fluorescence protein (GFP) fusion proteins is that intact recombinant protein can be quantified without the requirement of additional chemicals or modifications of the recombinant protein. This DSF assay can be performed using widely available equipment such as a real-time polymerase chain reaction (RT–PCR) instrument, microplates or microtubes, and fluorescent dye. This simple but powerful method can be easily applied in a wide range of research areas that require quantification of expressed recombinant proteins. [Copyright &y& Elsevier]

Highlights: [•] Optimization procedures for sample purification and characterization. [•] Rational design of buffers and additive screens compatible with Thermofluor assays. [•] Interpretation of thermal shift assays and melting-temperature midpoint calculations. [ABSTRACT FROM AUTHOR]

Abstract: Thermal shift analysis is becoming widely used as a method to identify initial hit ligands for inhibitor discovery or to identify ligands that may aid crystallization. The data analysis software provided by the equipment manufacturers or in the public domain is cumbersome to use. We have assessed a number of different approaches to generate a value for the melting temperature (T m) and implemented these methods in the program MTSA within the commercial software Matlab to provide an easy-to-use and rapid way to process experimental thermal shift data. The program outputs the T m, the quality of the fit, and the deviation from a standard value, the thermal shift ΔT m. Our analysis of these results includes a discussion of some issues with previous publications in this area. We conclude that the most suitable value for T m should be taken from the midpoint determined for a curve fitted to the experimental data with a five-parameter equation. In addition, we found that different ranking of ligand binding can be obtained using the different techniques when screening for binding of weak ligands such as fragments. Therefore, the technique should be used with caution for such screening. [Copyright &y& Elsevier]

Abstract: Thermal shift methods such as differential scanning fluorimetry and differential static light scattering are widely used to identify stabilizing conditions for proteins that might promote crystallization. Here we report a comparison of the two methods when applied to optimization of buffer conditions for protein–protein complexes. Most of the protein complexes under study were amenable to analysis using these two techniques. Protein complexes behave towards thermal denaturation in a manner similar to single proteins, showing a more or less sharp transition consistent with a two-state model of unfolding. A comparison of the melting and aggregation temperatures for single components and the reconstituted complexes can provide additional evidence for complex formation and can be used to identify buffer conditions in which protein–protein complex formation is favored. [Copyright &y& Elsevier]

Abstract: Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (T m) of 39.3±1.0 and 37.1±0.7°C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35°C over 120min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔT mmax =8°C, half-maximal effective ligand concentration, AC50 =3μM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in T m of ∼16°C, followed by MeCbl at ∼13°C, each evaluated at 50μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi>OHCbl>CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3μM, which compares well with the K d of 6μM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (ΔT mmax =4°C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7μM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease. [Copyright &y& Elsevier]

Abstract: Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent. While the wild-type protein was strictly specific for short-chain triglycerides (C4) in the temperature range 10–50 °C and displayed highest activity in the cold, its stabilized variant was able to accept C8 and C12 molecules and its selectivity was temperature dependent. We could not detect any improvement in the overall structural robustness of the mutant when the structure was challenged by temperature or chemical denaturants. There is, however, strong evidence for local stabilization effects in the active-site region provided by two independent approaches. Differential scanning fluorimetry revealed that the exposure of hydrophobic patches (as the active site is) precedes denaturation, and molecular dynamics simulations confirmed that stability was obtained by restriction of the mobility of the lid, a flexible structure that regulates the access to the enzyme active site and influences its stability. This reduction of lid movements is suggested to be accompanied by a concomitant increase in the mobility of other protein regions, thus accounting for the observed broadening of substrate specificity. [Copyright &y& Elsevier]

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the K d values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT–PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL l -cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT–PCR machines in a hydrophilic or hydrophobic environment. [ABSTRACT FROM AUTHOR]

[Display omitted] • Screening of a compound library against TFIIH subunit p8 has been carried out. • Interactions of 8 compounds with p8 were identified using STD-NMR spectroscopy. • Molecular docking showed interactions of compounds with hydrophobic core of p8. • In DSF assay compounds destabilize the P8 by negatively shifting the Tm (≥ 2°C). • These compounds were found non-cytotoxic against human fibroblast cell line (BJ). The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the β3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents. [ABSTRACT FROM AUTHOR]

TRF2 is a telomere associated protein which plays an important role in telomere maintenance. Knockdown of TRF2 can cause chromosomal end to end fusions and induce DNA damage responses. TRF2 exerts its functions partially by recruiting a number of accessory proteins through its TRF homology domain (TRFH), therefore identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions of TRF2 with its associated proteins is important to elucidate the molecular mechanism of these protein-protein interactions. Development of robust and sensitive screening and evaluation assays is critical to the identification of TRF2 inhibitors, in this paper we reported the development and optimization of a cascade of screening and binding affinity evaluation assays, including a competitive FP (Fluorescence Polarization) assay utilized in our previous research, and two novel label-free DSF (Differential Scanning Fluorescence) and BLI (Biolayer Interferometry) assays. A previously identified TRF2 inhibitor TRF2-27 was used as an internal reference compound and evaluated in all of these assays. According to the results, DSF assay is not suitable for TRF2 screening because of the low ΔT m , while the optimized labeled-free BLI assay was demonstrated to be an accurate and reproducible assay for TRF2 inhibitor screening and characterization. Image 1 • Three assays for the evaluation of binding affinities of TRF2 inhibitors were explored. • 1:1 kinetic binding sensorgrams were obtained from label-free BioLayer Interferometry (BLI). • K d values of the reference compound from BLI assays consist with K i values from competitive FP assays. [ABSTRACT FROM AUTHOR]

Among the biological approaches to therapeutics, are the cells, such as CAR-T cells engineered or not, the antibodies armed or not, and the smaller protein scaffolds that can be modified to render them specific of other proteins, à la façon of antibodies. For several years, we explored ways to substitute antibodies by nanobodies (also known as VHHs), the smallest recognizing part of camelids' heavy-chain antibodies: production of those small proteins in host microorganisms, minute analyses, characterization, and qualification of their affinity towards designed targets. Here, we present three standard VHHs described in the literature: anti-albumin, anti-EGF receptor and anti-HER2, a typical cancer cell surface -associated protein. Because they differ slightly in global structure, they are good models to assess our body of analytical methodologies. The VHHs were expressed in several bacteria strains in order to identify and overcome the bottlenecks to obtain homogeneous preparations of this protein. A large panel of biophysical tools, ranging from spectroscopy to mass spectrometry, was here combined to assess VHH structural features and the impact of the disulfide bond. The routes are now ready to move to more complex VHHs raised against specific targets in numerous areas including oncology. Image 1 • VHH are small antibody-like proteins. They are entering the therapeutic arsenal. • VHH purity, homogeneity, structure and affinity towards their targets are described. • An analytical workflow to qualify those proteins is presented to validate the molecules. [ABSTRACT FROM AUTHOR]