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Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.
Isothermal titration calorimetry (ITC) is the only technique able to determine both the enthalpy and entropy of noncovalent association in a single experiment. The standard data analysis method based on nonlinear regression, however, provides unrealistically small uncertainty estimates due to its neglect of dominant sources of error. Here, we present a Bayesian framework for sampling from the posterior distribution of all thermodynamic parameters and other quantities of interest from one or more ITC experiments, allowing uncertainties and correlations to be quantitatively assessed. For a series of ITC measurements on metal:chelator and protein:ligand systems, the Bayesian approach yields uncertainties which represent the variability from experiment to experiment more accurately than the standard data analysis. In some datasets, the median enthalpy of binding is shifted by as much as 1.5 kcal/mol. A Python implementation suitable for analysis of data generated by MicroCal instruments (and adaptable to other calorimeters) is freely available online.
Some of the best nucleating agents in nature are ice-nucleating proteins, which boost ice growth better than any other material. They can induce immersion freezing of supercooled water only a few degrees below 0 °C. An open question is whether this ability also extends to the deposition mode, i.e., to water vapor. In this work, we used three proteins, apoferritin, InaZ (ice nucleation active protein Z), and myoglobin, of which the first two are classified as ice-nucleating proteins for the immersion freezing mode. We studied the ice nucleation ability of these proteins by differential scanning calorimetry (immersion freezing) and by environmental scanning electron microscopy (deposition freezing). Our data show that InaZ crystallizes water directly from the vapor phase, while apoferritin first condenses water in the supercooled state, and subsequently crystallizes it, just as myoglobin, which is unable to nucleate ice.
We report a web-based tool for analysis of experiments using indirect calorimetry to measure physiological energy balance. CalR simplifies the process to import raw data files, generate plots, and determine the most appropriate statistical tests for interpretation. Analysis using the generalized linear model (which includes ANOVA and ANCOVA) allows for flexibility in interpreting diverse experimental designs, including those of obesity and thermogenesis. Users also may produce standardized output files for an experiment that can be shared and subsequently re-evaluated using CalR. This framework will provide the transparency necessary to enhance consistency, rigor, and reproducibility. The CalR analysis software will greatly increase the speed and efficiency with which metabolic experiments can be organized, analyzed per accepted norms, and reproduced and will likely become a standard tool for the field. CalR is accessible at https://CalRapp.org/.
Hydrolysis of β-lactam antibiotic by β-lactamase is the most common mechanism of β-lactam resistance in clinical isolates. Timely detection and characterization of β-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on β-lactamases.
We present a strategy for carrying out high-precision calculations of binding free energy and binding enthalpy values from molecular dynamics simulations with explicit solvent. The approach is used to calculate the thermodynamic profiles for binding of nine small molecule guests to either the cucurbit[7]uril (CB7) or β-cyclodextrin (βCD) host. For these systems, calculations using commodity hardware can yield binding free energy and binding enthalpy values with a precision of ∼0.5 kcal/mol (95% CI) in a matter of days. Crucially, the self-consistency of the approach is established by calculating the binding enthalpy directly, via end point potential energy calculations, and indirectly, via the temperature dependence of the binding free energy, i.e., by the van't Hoff equation. Excellent agreement between the direct and van't Hoff methods is demonstrated for both host-guest systems and an ion-pair model system for which particularly well-converged results are attainable. Additionally, we find that hydrogen mass repartitioning allows marked acceleration of the calculations with no discernible cost in precision or accuracy. Finally, we provide guidance for accurately assessing numerical uncertainty of the results in settings where complex correlations in the time series can pose challenges to statistical analysis. The routine nature and high precision of these binding calculations opens the possibility of including measured binding thermodynamics as target data in force field optimization so that simulations may be used to reliably interpret experimental data and guide molecular design.
Although drug development typically focuses on binding thermodynamics, recent studies suggest that kinetic properties can strongly impact a drug candidate's efficacy. Robust techniques for measuring inhibitor association and dissociation rates are therefore essential. To address this need, we have developed a pair of complementary isothermal titration calorimetry (ITC) techniques for measuring the kinetics of enzyme inhibition. The advantages of ITC over standard techniques include speed, generality, and versatility; ITC also measures the rate of catalysis directly, making it ideal for quantifying rapid, inhibitor-dependent changes in enzyme activity. We used our methods to study the reversible covalent and non-covalent inhibitors of prolyl oligopeptidase (POP). We extracted kinetics spanning three orders of magnitude, including those too rapid for standard methods, and measured sub-nM binding affinities below the typical ITC limit. These results shed light on the inhibition of POP and demonstrate the general utility of ITC-based enzyme inhibition kinetic measurements.
Studies of protein unfolding mechanisms are critical for understanding protein functions inside cells, de novo protein design as well as defining the role of protein misfolding in neurodegenerative disorders. Calorimetry has proven indispensable in this regard for recording full energetic profiles of protein unfolding and permitting data fitting based on unfolding pathway models. While both kinetic and thermodynamic protein stability are analysed by varying scan rates and reheating, the latter is rarely used in curve-fitting, leading to a significant loss of information from experiments. To extract this information, we propose fitting both first and second scans simultaneously. Four most common single-peak transition models are considered: (i) fully reversible, (ii) fully irreversible, (iii) partially reversible transitions, and (iv) general three-state models. The method is validated using calorimetry data for chicken egg lysozyme, mutated Protein A, three wild-types of haloalkane dehalogenases, and a mutant stabilized by protein engineering. We show that modelling of reheating increases the precision of determination of unfolding mechanisms, free energies, temperatures, and heat capacity differences. Moreover, this modelling indicates whether alternative refolding pathways might occur upon cooling. The Matlab-based data fitting software tool and its user guide are provided as a supplement.
The ability to characterize the development of metabolic function in neonatal rodents has been limited due to technological constraints. Low respiratory volumes and flows at rest pose unique problems, making it difficult to reliably measure O(2) consumption, CO(2) production, respiratory quotient (RQ), and energy expenditure (EE). Our aim was to develop and validate a commercial-grade indirect calorimetry system capable of characterizing the metabolic phenotype of individual neonatal rodents.
Bayesian regression is performed to infer parameters of thermodynamic binding models from isothermal titration calorimetry measurements in which the titrant is an enantiomeric mixture. For some measurements the posterior density is multimodal, indicating that additional data with a different protocol are required to uniquely determine the parameters. Models of increasing complexity-two-component binding, racemic mixture, and enantiomeric mixture-are compared using model selection criteria. To precisely estimate one of these criteria, the Bayes factor, a variation of bridge sampling is developed.
Recently, there has been a noticeable increase in the applications of composite mixtures containing molten salt and solid oxide for thermal energy conversion and storage systems. This highlights that thermal conductivity of the composites are central for the purpose of designing and devising processes. Measuring the thermal conductivity of molten samples at elevated temperatures remains challenging. In this study, the possibility to use heat flux differential scanning calorimetry (DSC) to measure the thermal conductivity of molten samples at elevated temperatures is reported for the first time. The thermal conductivity of composite mixtures containing eutectic (Li,Na)2CO3 with and without selected solid oxides at ~675 °C was determined by using the proposed DSC approach. This mixture is a candidate for high temperature waste heat conversion to electric energy. In the DSC measurement program, steps with repeated thermal cycles between 410 and 515 °C were included to limit the effect of the interface thermal contact resistance. The determined values 0.826 ± 0.001, and 0.077 ± 0.004 W m-1K-1 for the carbonate mixtures with and without solid MgO were found to match the reliable analysis at similar conditions.
We used microsecond time scale molecular dynamics simulations to compute, at high precision, binding enthalpies for cucurbit[7]uril (CB7) with eight guests in aqueous solution. The results correlate well with experimental data from previously published isothermal titration calorimetry studies, and decomposition of the computed binding enthalpies by interaction type provides plausible mechanistic insights. Thus, dispersion interactions appear to play a key role in stabilizing these complexes, due at least in part to the fact that their packing density is greater than that of water. On the other hand, strongly favorable Coulombic interactions between the host and guests are compensated by unfavorable solvent contributions, leaving relatively modest electrostatic contributions to the binding enthalpies. The better steric fit of the aliphatic guests into the circular host appears to explain why their binding enthalpies tend to be more favorable than those of the more planar aromatic guests. The present calculations also bear on the validity of the simulation force field. Somewhat unexpectedly, the TIP3P water yields better agreement with experiment than the TIP4P-Ew water model, although the latter is known to replicate the properties of pure water more accurately. More broadly, the present results demonstrate the potential for computational calorimetry to provide atomistic explanations for thermodynamic observations.
Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and β-lactoglobulin to Toyopearl Butyl-650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δhads , was observed to be dependent on temperature and salt concentration. Δhads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δhads for β-lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δhads increased with increasing temperature. The influence of salt concentration on Δhads was also more pronounced for bovine serum albumin than for β-lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques.
Interactions between charges play a role in protein stability and contribute to the energetics of binding between various charged ligands. Ionic surfactants are charged molecules, whose interactions with proteins are still rather poorly understood despite their wide applications. Here, we show by isothermal titration calorimetry that cationic alkylammonium surfactants bind to negatively charged polyaspartate and polyglutamate homopolymers stoichiometrically, i.e., one surfactant molecule per charged amino acid. Similarly, negatively charged alkyl sulfates (e.g., sodium dodecyl sulfate) and alkane sulfonates bind stoichiometrically to positively charged polylysine, polyornithine, and polyarginine homopolymers. In these reactions, the interacting counterparts form ion pairs and the resulting electrostatically neutral complex coprecipitates from solution. The enthalpies and heat capacities are determined for various pairs of ionic surfactants and charged amino acid homopolymers. These results show the energetic contributions of ionic headgroups and the CH2 group to surfactant interactions with proteins.
Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).1.
Thermal denaturation of Escherichia coli maltodextrin glucosidase was studied by differential scanning calorimetry, circular dichroism (230 nm), and UV-absorption measurements (340 nm), which were respectively used to monitor heat absorption, conformational unfolding, and the production of solution turbidity. The denaturation was irreversible, and the thermal transition recorded at scan rates of 0.5-1.5 K/min was significantly scan-rate dependent, indicating that the thermal denaturation was kinetically controlled. The absence of a protein-concentration effect on the thermal transition indicated that the denaturation was rate-limited by a mono-molecular process. From the analysis of the calorimetric thermograms, a one-step irreversible model well represented the thermal denaturation of the protein. The calorimetrically observed thermal transitions showed excellent coincidence with the turbidity transitions monitored by UV-absorption as well as with the unfolding transitions monitored by circular dichroism. The thermal denaturation of the protein was thus rate-limited by conformational unfolding, which was followed by a rapid irreversible formation of aggregates that produced the solution turbidity. It is thus important to note that the absence of the protein-concentration effect on the irreversible thermal denaturation does not necessarily means the absence of protein aggregation itself. The turbidity measurements together with differential scanning calorimetry in the irreversible thermal denaturation of the protein provided a very effective approach for understanding the mechanisms of the irreversible denaturation. The Arrhenius-equation parameters obtained from analysis of the thermal denaturation were compared with those of other proteins that have been reported to show the one-step irreversible thermal denaturation. Maltodextrin glucosidase had sufficiently high kinetic stability with a half-life of 68 days at a physiological temperature (37°C).
Substrate binding properties of the large (LS) and small (SS) subunits of potato tuber ADP-glucose pyrophosphorylase were investigated by using isothermal titration calorimetry. Our results clearly show that the wild type heterotetramer (S(WT)L(WT)) possesses two distinct types of ATP binding sites, whereas the homotetrameric LS and SS variant forms only exhibited properties of one of the two binding sites. The wild type enzyme also exhibited significantly increased affinity to this substrate compared to the homotetrameric enzyme forms. No stable binding was evident for the second substrate, glucose-1-phosphate, in the presence or absence of ATPγS suggesting that interaction of glucose-1-phosphate is dependent on hydrolysis of ATP and supports the Theorell-Chance bi bi reaction mechanism.
Here, we present a protocol for assessing metabolic activity of bacterial populations by measuring heat flow using isothermal calorimetry. We outline the steps for preparing the different growth models of Pseudomonas aeruginosa and performing continuous metabolic activity measurements in the calScreener. We detail simple principal component analysis to differentiate between metabolic states of different populations and probabilistic logistic classification to assess resemblance to wild-type bacteria. This protocol for fine-scale metabolic measurement can aid in understanding microbial physiology. For complete details on the use and execution of this protocol, please refer to Lichtenberg et al. (2022).1.
The use of indirect calorimetry (IC) is increasing due to its precision in resting energy expenditure (REE) measurement in critically ill patients. Thus, we aimed to evaluate the clinical outcomes of an IC-guided nutrition therapy compared to predictive equations strategy in such a patient population.
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