Name: measuring enzymatic stability by isothermal titration calorimetry
Uploaded: 2019-03-26T14:30:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Measuring Enzymatic Stability by Isothermal Titration Calorimetry at JoVE.com

1. Preparing samples
<ol>
	<li>1,000 mL of 0.1 M sodium acetate buffer at pH 4.6
	<ol>
		<li>Measure 800 mL of distilled water in a 1,000 mL graduated beaker.</li>
		<li>Weigh 8.2 g of anhydrous sodium acetate and add it to the beaker.</li>
		<li>Place the beaker on a stir plate, place a stir rod into the beaker, turn on the stir plate and stir until completely dissolved.</li>
		<li>When the anhydrous sodium acetate is completely dissolved, measure the pH of the solution with a calibrated pH meter.</li>
		<li>Add 1 M HCl or NaOH accordingly to obtain the desired pH 4.6.</li>
		<li>Add distilled water until the total volume is 1,000 mL.</li>
		<li>Store at room temperature until use.</li>
	</ol>
	</li>
	<li>Enzyme solution
	<ol>
		<li>Prepare 10 mL of the enzyme solution within the 10-30 mg/mL range by first measuring 8 mL of the 0.1 M sodium acetate buffer pH 4.6 in a 15 mL graduated cylinder.&#160;</li>
		<li>Add the buffer solution into a 15 mL conical tube with enzyme and shake vigorously until the enzyme has dissolved.</li>
		<li>Add more buffer solution until the total volume is 10 mL.</li>
		<li>Store the enzyme solution at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Substrate solution
	<ol>
		<li>To prepare a substrate solution within the 300-600 mM range, calculate the amount of substrate needed in grams to make the desired concentration.</li>
		<li>Weigh out the substrate and place into a 100 mL glass beaker</li>
		<li>Measure 20 mL of the buffer solution using a 25 mL graduated cylinder and then add it to the glass beaker.</li>
		<li>Place the beaker on a stir plate and place a magnetic stir rod into the beaker. Turn on the heat and adjust the stirring speed accordingly.</li>
		<li>Allow stirring to continue until the substrate has dissolved.</li>
		<li>Pour the substrate solution into a 50 mL conical tube and add 0.1 M sodium acetate buffer pH 4.6 until the total volume is 45 mL. Mix by shaking.</li>
		<li>Store the substrate solution at room temperature until use.</li>
	</ol>
	</li>
</ol>
2. Performing the experiment
<ol>
	<li>Preparing the ITC instrument&#160;
	<ol>
		<li>Ensure that the reference cell is loaded with 350 &#181;L of distilled water. Before loading the enzyme into the sample cell, verify that the sample cell has been cleaned.</li>
		<li>Cleaning protocol- Fill the loading syringe with 500 &#181;L of 2% cleaning solution (Table of Materials), carefully insert the needle into the sample cell, fill the cell and slowly remove the liquid using the same syringe. Dispose the liquid into a beaker. Repeat this step twice with 2% cleaning solution (Table of Materials), three times with 70% ethanol and then wash ten times with distilled water.</li>
		<li>Fill the loading syringe with 450 &#181;L of enzyme solution, carefully insert the needle all the way to the bottom of the sample cell and press the plunger down to the 100 &#181;L line slowly to prevent formation of air bubbles.</li>
		<li>Wash the 50 &#181;L titration syringe with distilled water three times by placing the needle tip into water then slowly taking up the water into the syringe, then dispensing the water into a waste container.</li>
		<li>Remove residual water by rinsing with substrate solution three times.</li>
		<li>Fill the titration syringe with substrate solution by drawing the solution up until the syringe is full without any air bubbles.&#160;</li>
		<li>With the syringe still in the substrate solution, remove the plunger and allow approximately 2 &#181;L of air to enter the top of the syringe and reinsert the plunger.</li>
		<li>Remove the buret handle of the ITC, place the syringe inside the buret handle and screw until tight.&#160;</li>
		<li>Wipe the tip of the stirrer with a lint-free tissue, then carefully place the buret handle into the ITC instrument and lock it in place.</li>
	</ol>
	</li>
</ol>
3. Setting up ITCrun&#160;
<ol>
	<li>On the computer, open ITCrun and click Set up.</li>
	<li>Click Stirring rate and set to 350 RPM. Check the syringe size (&#181;L) and ensure it is at 50 &#181;L.</li>
	<li>Set the temperature and press Update. It is recommended that this step be performed at least 1 h before preparing the ITC. This allows enough time for the instrument to heat up or cool down as needed.</li>
	<li>For the experiment setup, select incremental titration.</li>
	<li>Click Insert to setup the injections. Adjust the injection interval to 5,400 s, injection volume (&#181;L) to 4 and number of injections to 4. Press OK to confirm settings.</li>
	<li>In the Equilibration box, select Auto-equilibrate and Large expected heats. (If the expected heats are small, one can select Small under expected heats; however, this will increase the equilibration time.)</li>
	<li>Set the initial baseline to 300 s.</li>
	<li>To start the run, click the Start symbol next to Stirring rate and then click Start which is located next to the wrench symbol.</li>
	<li>Save the file and allow the instrument to run.</li>
</ol>
4. Analyzing data
<ol>
	<li>Open the file in NanoAnalyze. Click Data and select Data columns.</li>
	<li>Select all data, copy and then paste the data into Microsoft Excel.</li>
	<li>Adjust zero baseline by adding the value required at 300 s to make it zero. Apply this correction to the entire column of heat rate values.&#160;</li>
	<li>Find the minimum or maximum value of the heat rate for each injection by using the equation: =MIN(cell:cell) or MAX(cell:cell). Each data point represents the peak enzymatic activity of the enzyme at each injection.</li>
	<li>Plot graphs of the MIN or MAX values against the time at which the value occurred during the titration.</li>
</ol>

<ol>
	<li>Anastas, P., Eghbali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+chemistry:+principles+and+practice.">Green chemistry: principles and practice.</a> Chemical Society Reviews. 39 (1), 301-312 (2010).</li><li>Jin, L. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+Recombinant+Glucose+Isomerase+for+Efficient+Production+of+High+Fructose+Corn+Syrup.">Immobilization of Recombinant Glucose Isomerase for Efficient Production of High Fructose Corn Syrup.</a> Applied Biochemistry Biotechnoiogy. 183 (1), 293-306 (2017).</li><li>Budak, &. #. 3. 5. 0. ;. &. #. 2. 1. 4. ;., Ko&#231;ak, C., Bron, P. A., de Vries, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbial Cultures and Enzymes Dairy Technology. , 182-203 (2018).</li><li>van Donkelaar, L. H. G., Mostert, J., Zisopoulos, F. K., Boom, R. M., van der Goot, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+enzymes+for+beer+brewing:+Thermodynamic+comparison+on+resource+use.">The use of enzymes for beer brewing: Thermodynamic comparison on resource use.</a> Energy. 115, 519-527 (2016).</li><li>Rodriguez-Colinas, B., Fernandez-Arrojo, L., Ballesteros, A. O., Plou, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galactooligosaccharides+formation+during+enzymatic+hydrolysis+of+lactose:+Towards+a+prebiotic-enriched+milk.">Galactooligosaccharides formation during enzymatic hydrolysis of lactose: Towards a prebiotic-enriched milk.</a> Food Chemistry. 145, 388-394 (2014).</li><li>Lawrence, P. B., Price, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+PEGylation+influences+protein+conformational+stability.">How PEGylation influences protein conformational stability.</a> Current Opinions in Chemical Biology. 34, 88-94 (2016).</li><li>Bernal, C., Rodriguez, K., Martinez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+enzyme+immobilization+and+protein+engineering:+An+alternative+path+for+the+development+of+novel+and+improved+industrial+biocatalysts.">Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts.</a> Biotechnology Advances. 36 (5), 1470-1480 (2018).</li><li>Rigoldi, F., Donini, S., Redaelli, A., Parisini, E., Gautieri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Engineering+of+thermostable+enzymes+for+industrial+applications.">Review: Engineering of thermostable enzymes for industrial applications.</a> Applied Bioengeneering. 2 (1), 011501 (2018).</li><li>Johnson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+scanning+calorimetry+as+a+tool+for+protein+folding+and+stability.">Differential scanning calorimetry as a tool for protein folding and stability.</a> Archives of Biochemistry and Biophysics. 531 (1), 100-109 (2013).</li><li>Chen, N. G., Gregory, K., Sun, Y., Golovlev, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+model+of+thermal+deactivation+of+enzymes.">Transient model of thermal deactivation of enzymes.</a> Biochimica et biophysica acta. 1814 (10), 1318-1324 (2011).</li><li>Mason, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorimetric+Methods+for+Measuring+Stability+and+Reusability+of+Membrane+Immobilized+Enzymes.">Calorimetric Methods for Measuring Stability and Reusability of Membrane Immobilized Enzymes.</a> Jounal of Food Science. , (2017).</li><li>Leksmono, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Lactase+Enzymatic+Activity+in+the+Teaching+Lab.">Measuring Lactase Enzymatic Activity in the Teaching Lab.</a> Journal of visualized experiments : Journal of Visual Experiments. (138), e54377 (2018).</li></ol>

A major advantage of the ITC enzyme stability assay described here is automation. Once all the appropriate buffers and solutions are made, the set-up time for each assay is approximately 15 min for the person doing the assay. In contrast, the conventional assays for invertase and lactase activity require about 2 h with continual involvement of the person doing the assay and many enzymatic activity assays take considerably more person-hours. In a previous publication, we have demonstrated how data from the ITC method comp...

Enzymes are proteins capable of catalyzing a wide array of organic reactions. Most enzymes function in aqueous solution at near neutral pH thus avoiding the use of harsh solvents. Because of their high selectivity, enzyme catalyzed reactions produce fewer (in some cases no byproducts) byproducts than non-selective catalysts such as acids and bases1. This is especially relevant in food manufacturing where all chemical reactions must be done so the final product is safe for human consumption. Currently, enzymes are used to produce high fructose corn syrup2, cheese3, beer4, lactose-free milk5, and other important food products. While this paper focuses on enzyme use in the food industry, there are many other uses for enzymes including in green chemistry and drug synthesis.&#160;
The utility of enzymes is limited by the stability of enzyme activity, which depends on maintaining the three-dimensional structure of the enzyme. The enzyme structure can be stabilized by modifications such as PEGylation6, immobilization on a solid support7, genetic modifications8, and formulations. Currently, enzyme stability is typically measured by differential scanning calorimetry (DSC) and endpoint enzyme activity assays9. DSC measures the temperature at which an enzyme unfolds; the higher the temperature, the more stable the structure. However, loss of activity often occurs at a lower temperature than required to unfold the enzyme or domains within the enzyme10. Therefore, DSC is not sufficient to determine whether an enzyme modification increases the stability of enzyme activity. Endpoint enzyme assays are usually time intensive, require multiple samples, and often involve a coupled colorimetric reaction that is not applicable to highly colored or opaque solutions or suspensions.
This work demonstrates a method for direct measurement of the stability of enzyme activity by isothermal titration calorimetry (ITC). ITC measures the rate of heat released or absorbed during the course of a reaction. Since nearly all reactions produce or absorb heat, ITC can be used for most enzyme-catalyzed reactions, including reactions that do not have a coupled reaction or occur in opaque media such as milk. ITC has been used for many decades to measure chemical kinetic parameters for many kinds of reactions, but the protocol presented here focuses on using ITC to measure the peak heat rate of enzyme-catalyzed reactions and demonstrates that enzyme activity is linearly correlated with the peak heat rate. ITC measurements of peak heat rates are mostly autonomous and require very little personnel time to setup and analyze.&#160;

<table><tbody><tr><td>a-Lactose</td><td>Fisher Scientific&amp;nbsp;</td><td>unknown (too old)</td><td>500g</td></tr><tr><td>Sodium Acetate, Anhydrous 99% min</td><td>Alfa Aesar</td><td>A13184-30</td><td>250g</td></tr><tr><td>Lactase&amp;nbsp;</td><td>MP Bio</td><td>100780</td><td>5g</td></tr><tr><td>Hydrocholric Acid Solution, 1N&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>SA48-500</td><td>500mL</td></tr><tr><td>Benchtop Meter- pH</td><td>VWR</td><td>89231-622</td><td></td></tr><tr><td>Ethanol 70%</td><td>Fisher Scientific&amp;nbsp;</td><td>BP8231GAL</td><td>1gallon</td></tr><tr><td>Micro-90</td><td>Fisher Scientific&amp;nbsp;</td><td>NC024628</td><td>1L (cleaning solution)</td></tr></tbody></table>

measuring enzymatic stability by isothermal titration calorimetry

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.&#160; &#160;

The representative results in Figure 1 and Figure 5 show data from two enzymes, lactase and invertase. Lactase and invertase catalyze the hydrolysis of a disaccharide into two monosaccharides, endothermically and exothermically, respectively. Both enzymatic reactions were run at concentrations that precluded saturation of the enzyme.&#160;
The lactase data demonstrate how ITC data can be used to estimate enzyme stability. Four sequent...

Watch this Scientific Journal Video about Measuring Enzymatic Stability by Isothermal Titration Calorimetry at JoVE.com

Measuring Enzymatic Stability by Isothermal Titration Calorimetry

The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed ...

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Research

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13.0K Views.  Brigham Young University. The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme activity.

Video: Measuring Enzymatic Stability by Isothermal Titration Calorimetry

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Chemistry

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Biochemistry

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (kcat, KM, &#916;H) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.
In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (&#916;Hint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To evaluate the potential biotechnical value of these proteins, the analysis of protein structures and functions is a central challenge in this program. The stability of protein sample is essential to provide meaningful assay results and increase the crystallizability of the targets. The thermal shift assay (TSA), a fluorescence-based technique, is established as a popular method for optimizing the conditions for protein stability in high-throughput. In TSAs, the employed fluorophores are extrinsic, environmentally-sensitive dyes. An alternative, similar technique is nano differential scanning fluorimetry (nanoDSF), which relies on protein native fluorescence. We present here a novel osmolyte screen, a 96-condition screen of organic additives designed to guide crystallization trials through preliminary TSA experiments. Together with previously-developed pH and salt screens, the set of three screens provides a comprehensive analysis of protein stability in a wide range of buffer systems and additives. The utility of the screens is demonstrated in the TSA and nanoDSF analysis of lysozyme and Protein X, a target protein of the Virus-X project.

A protocol is presented to rapidly test the thermal stability of proteins in a variety of conditions through thermal shift assays and nano differential scanning fluorimetry. Buffer systems, salts and additives, together comprising three unique stability screens, are assayed with proteins to identify suitable buffers for functional and structural studies.

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To ...

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

Biology

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

A general protocol for the use of isothermal titration calorimetry to monitor the binding thermodynamics for biological systems with moderate binding affinities is presented.

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction.  In biological ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Bioengineering

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

Measuring Enzymatic Stability by Isothermal Titration Calorimetry

Summary

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Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Measuring In Vitro ATPase Activity for Enzymatic Characterization