R. W. H. V was supported by the McGill Natural Sciences and Engineering Research Council of Canada (NSERC) Training Program in Bionanomachines. A. K. M. and P. E. J. were supported by NSERC grants 327028-09 (A. K. M) and 238562 (P. E. J.).

1. Sample Preparation
<ol>
	<li>Purify the desired biomolecule13. 
	NOTE: This protocol uses purchased cocaine-binding DNA aptamer MN4 after exchanging against 2 M NaCl three times followed by three rounds of deionized water using a centrifugal filter with a 3 kDa molecular weight cut-off membrane.</li>
	<li>Synthesize and purify, or purchase the desired thermolabile ligand13. 
	NOTE: MN4 binds the thermolabile ligand cocaine. MN4 also binds quinine, which is used as a negative control for ligand thermolability at these experimental temperatures.</li>
	<li>Prepare buffers for dialysis of the purified biomolecule and dissolution of ligands (20 mM sodium phosphate and 140 mM NaCl buffer, pH 7.4, for MN4 and the ligands used here).</li>
	<li>Dialyze the biomolecule against at least 2 L of buffer using dialysis tubing with 0.5 - 1.0 kDa cut-off.</li>
	<li>Filter the final buffer (referred to as working buffer) through a 0.2 &#181;m filter that has been thoroughly equilibrated with buffer.</li>
	<li>Weigh out the desired masses of the ligands and dissolve them in filtered working buffer. If the desired ligand concentrations require masses that are too small to accurately weigh, make a concentrated ligand stock solution (10x for example). 
	NOTE: It is crucial that all DSC experiments utilize the same working buffer for the sample and ligand, i.e., never perform an experiment where the ligand is dissolved in a different batch of working buffer than the biomolecule as this will cause buffer mismatch artifacts in the data.</li>
	<li>Filter the biomolecule stock solution through a 0.2 &#181;m filter that has been thoroughly equilibrated with working buffer.</li>
	<li>Determine the biomolecule concentration by absorbance measurements (260 nm for nucleic acids like MN4 and 280 nm for proteins).</li>
	<li>Store the prepared biomolecule and ligand in a 4 &#176;C refrigerator (suitable for MN4 and the ligands used here), or at -20 or -80 &#176;C if the biomolecule and ligands tolerate freezing and long-term storage is required. Degas the buffer, biomolecule, and ligand solutions in a table top degasser (see Table of Materials) prior to loading into the DSC. 
	NOTE: Degassing helps to prevent bubble formation in the DSC at higher temperatures. Bubbles cause signal artifacts that obscure DSC peak shapes and baselines.</li>
</ol>
2. DSC Preparation
<ol>
	<li>Unscrew the pressure handle from the DSC (see Table of Materials).</li>
	<li>Run silicon tubing from the working buffer and attach it to the front flange (metal opening) of the reference capillary.</li>
	<li>Create a bridge between the reference and sample capillaries by connecting the rear reference flange to the front sample flange.</li>
	<li>Attach a piece of silicon tubing to the rear sample flange that runs to a waste flask with a vacuum line attached.</li>
	<li>Turn on the vacuum line to flush the DSC with 200 mL of working buffer.</li>
	<li>Load the reference capillary of the DSC with working buffer. Attach roughly 3 - 5 cm sections of silicon tubing to the reference capillary flanges.</li>
	<li>Insert a 1 mL pipette tip into the rear flange&#39;s silicon tubing. Draw 0.8 mL of working buffer with a pipette and insert the pipette tip with buffer into the front reference flange&#39;s silicon tubing.</li>
	<li>Gently press the pipette plunger down to pass the working buffer through the front silicon tubing into the reference capillary and up into the rear flange&#39;s attached pipette tip. Press down the pipette plunger until the working buffer level reaches just above the front silicon tubing, then release the pipette plunger until the working buffer level reaches just above the rear silicon tubing.
	<ol>
		<li>Repeat passing the working buffer back and forth to purge the volume in the reference capillary of bubbles. 
		NOTE: Usually, 10 passes of the solution back and forth are sufficient to clear any bubbles.</li>
	</ol>
	</li>
	<li>Cap the rear pipette tip with the thumb and gently pull up on the rear pipette tip and front pipette to remove them from the reference flanges with the silicon tubing attached.</li>
	<li>Load the sample capillary with working buffer as in steps 2.6 - 2.9. Place a black plastic cap on the rear reference and sample flanges, leaving the front flanges uncovered.</li>
	<li>Attach the pressure handle.</li>
	<li>Open the DSC software (see Table of Materials) and pressurize the instrument by clicking the red up arrow at the top of the interface once the power reading has stabilized; the DSC power is indicated in a box at the top right of the interface along with the instrument temperature and pressure reading. 
	NOTE: Monitor the power reading as the DSC pressurizes. Changes in power of more than ~ 10 &#181;W indicate bubble formation in the capillaries, which can cause artifacts in the data. The solutions must be removed and degassed further before continuing.</li>
	<li>Equilibrate the DSC with working buffer by performing a forward and reverse scan. In the &#34;Experimental Method&#34; tab on the left side of the screen, ensure that the &#34;Scanning&#34; option is selected to run the DSC in temperature scanning mode. 
	NOTE: Experimental parameters are the temperature scanning range, scan rate, low and high temperature equilibration time, and number of scans.
	<ol>
		<li>In the &#34;Temperature Parameters&#34; inset under the &#34;Experimental Method&#34; tab, click the button for &#34;Heating&#34;. Enter 1 and 100 &#176;C for the lower and upper experimental temperatures, 1 &#176;C/min for the scan rate, and 60 s for the equilibration period.</li>
		<li>Click the &#34;Add Series&#34; button under the input field for the equilibration period. Enter 2 into the &#34;Steps to add&#34; field in the popup window (for one heating and one cooling scan) and check the &#34;Alternate Heating/Cooling&#34; box. Click &#34;OK&#34;; the added scans appear in the lower portion of the interface. Check that the parameters for each scan are as desired.</li>
		<li>Start the experiment by clicking the green &#34;play&#34; button at the top of the interface. Navigate to the desired folder and input a file name for saving the experiment in the popup window. View the experiment progress by clicking the &#34;Data&#34; tab to the right of the &#34;Experiment Method&#34; tab.</li>
	</ol>
	</li>
</ol>
3. Collecting Thermolabile Ligand DSC Datasets
NOTE: The minimal procedure consists of five experiments: buffer reference experiments with and without ligand (used for baseline subtraction, see Discussion), sample experiments with the free biomolecule, the ligand-bound biomolecule, and the ligand-bound biomolecule with a longer high temperature equilibration period.
<ol>
	<li>Run reference experiments for baseline subtraction of the sample data. Reload the DSC with working buffer in both capillaries and collect multiple forward and reverse scans over a suitable temperature range at 1 &#176;C min-1 with an upper (high temperature) equilibration time of 120 s.
	<ol>
		<li>Delete the previous buffer equilibration scans from the lower portion of the interface by highlighting each individually and clicking the red X to the middle right of the interface. Add the new scans by clicking the &#34;Add Series&#34; button, entering 20 in the field for &#34;Steps to add&#34;, and checking the &#34;Alternate Heating/Cooling&#34; box. Click OK and run the experiment by clicking the green play button as above.</li>
		<li>Repeat steps 3.1 - 3.1.1 with working buffer containing the desired concentration of ligand in both capillaries to obtain the reference experiments for the ligand (collect two separate experiments using 120 s and 600 s high temperature equilibration times respectively, to be used in acquiring the rate constant for thermolabile ligand conversion). 
		NOTE: The scan rate used here ensures that the biomolecule in subsequent experiments is at thermal equilibrium in the forward and reverse scans (see Discussion). Scan rates &#60; 0.1 - 0.2 &#176;C min-1 lead to noisy thermograms and are not applicable in DSC experiments. The temperatures should extend from well below the melting temperature of the free biomolecule to well above the melting temperature of ligand-saturated biomolecule (~ 20 - 80 &#176;C for MN4). Verify reproducibility of the scans (for example, 10 forward and 10 reverse scans for 20 total is sufficient).</li>
		<li>If using multiple ligands (such as cocaine and quinine), flush the DSC with 200 mL of working buffer (repeat from&#160;step 2.5) between runs in order to remove ligand from the capillaries and prevent cross-contamination. 
		NOTE: It is helpful to perform a replicate experiment on the free biomolecule after a ligand-bound run in order to check if the previous ligand adsorbs strongly to the capillary walls and is not adequately removed with buffer flushing. If the thermograms for the free biomolecule appear to be shifted to a larger magnitude and higher denaturation temperature after the ligand-bound experiment, it is likely that the previous ligand is still present in the calorimeter after flushing. Remove the adsorbed ligand by incubating the capillaries with 20% Contrad-70 for 1 h at 60 &#176;C with the plastic caps and pressure handle off. In the DSC software, change the experimental mode to &#34;Isothermal&#34; under the Experimental Method tab. Choose 3,600 s for the duration and 60 &#176;C for the isothermal temperature, with zero entered for the equilibration time. Click &#34;Add to Experimental Method&#34;. The isothermal experiment appears in the lower part of the screen. After completion, flush the instrument with 2 L deionized water and repeat from step 2.5.</li>
	</ol>
	</li>
	<li>Run sample experiments using the same DSC loading procedure and experimental parameters as the reference scans. For the free biomolecule data set, ensure that the reference capillary contains the working buffer while the sample capillary contains the free biomolecule at the desired concentration in working buffer.
	<ol>
		<li>For the ligand-bound experiments, ensure that the ligand is in the working buffer in the reference capillary, and the biomolecule plus ligand are in working buffer in the sample capillary. Flush the system between additions of different ligands as in step 2.5.</li>
	</ol>
	</li>
	<li>Perform one additional experiment with the biomolecule bound to the thermolabile ligand where the high temperature equilibration period is increased to 600 s and all other experimental parameters are the same as step 3.2.1. 
	NOTE: The duration of the high temperature equilibration period for the second ligand-bound experiment is simply chosen to ensure that the ligand is more rapidly depleted than the short equilibration time experiment. If the ligand-bound peaks from the first experiment decay slowly as a function of scan number (e.g., the differences in successive peak maxima are &#8804; 0.5 &#176;C), estimate 10- to 20-fold increases in the high temperature equilibration period in order to adequately deplete the ligand during the second experiment. The accurate calculation of the rate constant for ligand conversion requires that the second experiment has swifter depletion of the ligand relative to the first. The ligand concentrations extracted from the global analysis of the two experiments will be similar and therefore unusable if the ligand depletion is not sufficiently accelerated in the second experiment.</li>
</ol>
4. Data Processing
<ol>
	<li>Open the DSC experiment files in the DSC data analysis software (see Table of Materials) and export the raw power data as spreadsheets.</li>
	<li>Import the spreadsheets containing the raw power data into software for data fitting.</li>
	<li>Baseline subtract the sample data by subtracting the buffer power data from the free and ligand-bound biomolecule experiments. 
	NOTE: For the thermolabile ligand experiment, the concentration of initial ligand is decreasing with each scan. Therefore, it is ideal to subtract the buffer scan 1 from sample scan 1, and so on. We have found that the buffer scans with cocaine do not change appreciably as the ligand conversion proceeds and therefore a single thermolabile ligand buffer scan can be used to subtract all of the thermolabile ligand-bound data.</li>
	<li>Convert the baseline-subtracted sample power data to heat capacity. 
	NOTE: The conversion requires the biomolecule&#39;s partial specific volume, which can be estimated14,15,16. The equation for converting power to heat capacity has been described previously17.</li>
</ol>
5. Data Analysis
<ol>
	<li>Globally fit the short equilibration time thermolabile ligand-bound heat capacity dataset with a single set of baseline, ligand concentration, folding, and ligand binding parameters as described previously4.</li>
	<li>Repeat the global fit for the long equilibration time dataset in order to calculate the rate constant for thermolabile ligand conversion as described previously4.</li>
</ol>

<ol>
	<li>Bruylants, G., Wouters, J., Michaux, C. <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+in+life+science:+thermodynamics,+stability,+molecular+recognition+and+application+in+drug+design.">Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design.</a> Curr Med Chem. 12 (17), 2011-2020 (2005).</li><li>Privalov, P. L., Dragan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcalorimetry+of+biological+macromolecules.">Microcalorimetry of biological macromolecules.</a> Biophys Chem. 126 (1-3), 16-24 (2007).</li><li>Brandts, J. F., Lin, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+strong+to+ultratight+protein+interactions+using+differential+scanning+calorimetry.">Study of strong to ultratight protein interactions using differential scanning calorimetry.</a> Biochemistry. 29 (29), 6927-6940 (1990).</li><li>Harkness, R. W., Slavkovic, S., Johnson, P. E., Mittermaier, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+characterization+of+folding+and+binding+interactions+with+thermolabile+ligands+by+DSC.">Rapid characterization of folding and binding interactions with thermolabile ligands by DSC.</a> Chem Commun. 52 (92), 13471-13474 (2016).</li><li>Garbett, N. C., Chaires, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+studies+for+drug+design+and+screening.">Thermodynamic studies for drug design and screening.</a> Expert Opin Drug Dis. 7 (4), 299-314 (2012).</li><li>Holdgate, G. A., Ward, W. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+binding+thermodynamics+in+drug+discovery.">Measurements of binding thermodynamics in drug discovery.</a> Drug Discov Today. 10 (22), 1543-1550 (2005).</li><li>Plotnikov, V., 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=An+autosampling+differential+scanning+calorimeter+instrument+for+studying+molecular+interactions.">An autosampling differential scanning calorimeter instrument for studying molecular interactions.</a> Assay Drug Dev Technol. 1 (1), 83-90 (2002).</li><li>Schon, A., Lam, S. Y., Freire, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics-based+drug+design:+strategies+for+inhibiting+protein-protein+interactions.">Thermodynamics-based drug design: strategies for inhibiting protein-protein interactions.</a> Future Med Chem. 3 (9), 1129-1137 (2011).</li><li>Peri&#225;nez Parraga, L., G-L, A., Gam&#243;n Runnenberg, I., Seco Melantuche, R., Delgado S&#225;nchez, O., Puigvent&#243;s Latorre, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermolabile+Drugs.+Operating+Procedure+In+the+Event+of+Cold+Chain+Failure.">Thermolabile Drugs. Operating Procedure In the Event of Cold Chain Failure.</a> Farmacia Hospitalaria. 35 (4), 1-28 (2011).</li><li>Murray, J. B., Alshora, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+Cocaine+in+Aqueous-Solution.">Stability of Cocaine in Aqueous-Solution.</a> J Clin Pharmacy. 3 (1), 1-6 (1978).</li><li>Waterman, K. C., 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=Hydrolysis+in+pharmaceutical+formulations.">Hydrolysis in pharmaceutical formulations.</a> Pharm. Dev. Technol. 7 (2), 113-146 (2002).</li><li>Mergny, J. L., Lacroix, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thermal+melting+curves.">Analysis of thermal melting curves.</a> Oligonucleotides. 13 (6), 515-537 (2003).</li><li>Neves, M. A., Reinstein, O., Johnson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+stem+length-dependent+binding+mechanism+for+the+cocaine-binding+aptamer.+A+combined+NMR+and+calorimetry+study.">Defining a stem length-dependent binding mechanism for the cocaine-binding aptamer. A combined NMR and calorimetry study.</a> Biochemistry. 49 (39), 8478-8487 (2010).</li><li>Bonifacio, G. F., Brown, T., Conn, G. L., Lane, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+electrophoretic+and+hydrodynamic+properties+of+DNA+and+RNA+oligonucleotide+duplexes.">Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes.</a> Biophys J. 73 (3), 1532-1538 (1997).</li><li>Durchschlag, H., Hinz, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3.">Chapter 3.</a> Thermodynamic Data for Biochemistry and Biotechnology. , 45-128 (1986).</li><li>Hellman, L. M., Rodgers, D. W., Fried, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenomenological+partial-specific+volumes+for+G-quadruplex+DNAs.">Phenomenological partial-specific volumes for G-quadruplex DNAs.</a> Eur Biophys J Biophy. 39 (3), 389-396 (2010).</li><li>Farber, P., Darmawan, H., Sprules, T., Mittermaier, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Protein+Folding+Cooperativity+by+Differential+Scanning+Calorimetry+and+NMR+Spectroscopy.">Analyzing Protein Folding Cooperativity by Differential Scanning Calorimetry and NMR Spectroscopy.</a> J Am Chem Soc. 132 (17), 6214-6222 (2010).</li><li>Reinstein, O., 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=Quinine+binding+by+the+cocaine-binding+aptamer.+Thermodynamic+and+hydrodynamic+analysis+of+high-affinity+binding+of+an+off-target+ligand.">Quinine binding by the cocaine-binding aptamer. Thermodynamic and hydrodynamic analysis of high-affinity binding of an off-target ligand.</a> Biochemistry. 52 (48), 8652-8662 (2013).</li><li>Tellinghuisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+error+propagation.">Statistical error propagation.</a> J Phys Chem. A. 105 (15), 3917-3921 (2001).</li><li>Drobnak, I., Vesnaver, G., Lah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model-based+thermodynamic+analysis+of+reversible+unfolding+processes.">Model-based thermodynamic analysis of reversible unfolding processes.</a> J Phys Chem B. 114 (26), 8713-8722 (2010).</li></ol>

The authors declare no conflicts of interest.

Modifications and troubleshooting
The details of the global fitting analysis used in Figure 1 and Figure 2 have been described previously4. Here, we outline practical aspects of performing and analyzing DSC binding experiments with thermolabile ligands. Note that a DSC baseline obtained for the thermolabile ligand alone is subtracted from the ligand + biomolecule dataset, effectively cancelling ou...

Differential scanning calorimetry (DSC) is a powerful method for quantitating biomolecular binding and folding interactions1,2,3. The strengths of DSC include its ability to elucidate binding and folding mechanisms, and to yield the corresponding thermodynamic parameters2,3. Furthermore, DSC can be performed in solution under near-physiological conditions and does not require labeling of the biomolecule or ligand, e.g., with fluorophores, spin-labels or nuclear isotopes4. The instrument scans in temperature, measuring the amount of heat required to denature the biomolecule in the presence and absence of ligand. The resulting thermograms are used to extract the thermodynamic parameters governing the ligand binding and folding processes. The information provided by DSC or other thermodynamic techniques is critical to guiding the design of drugs targeting biomolecules1,5,6,7,8. However, the repeated scanning to high temperatures (~ 60 - 100 &#176;C) can be problematic. For example, many pharmaceutically important compounds undergo rearrangement or decomposition upon sustained exposure to high temperatures9,10,11, i.e., they are thermolabile. Examination of binding interactions by DSC typically requires multiple forward and reverse scans in order to verify the reproducibility of the thermogram for thermodynamic analyses12. Thermal conversion of an initial ligand to a secondary form with altered binding characteristics leads to pronounced differences in the shape and position of successive thermograms, since the concentration of the initial ligand decreases with each scan while the thermal conversion products accumulate. These datasets are not amenable to traditional analyses.
We have recently developed a global fitting method for thermolabile ligand DSC datasets that yields the complete set of thermodynamic parameters governing the biomolecular folding and binding interactions from a single ligand-bound experiment referenced to the requisite thermogram for the free biomolecule4. The analysis reduces the experimental time and sample required by ~ 10-fold compared to standard DSC approaches. We have accounted for ligand thermal conversion by assuming this happens during the high temperature portion of each scan where the thermogram does not depend on ligand concentration. Therefore, the ligand concentration is a constant within the portion of the thermogram that is used to extract thermodynamic parameters. We additionally demonstrated how the rate constant for ligand thermal conversion can be obtained by performing one supplementary experiment with a longer high temperature equilibration period. For systems where ligand thermal conversion is less temperature-dependent (i.e., occurring appreciably at all temperatures), the analysis can be modified to include variable ligand concentrations. Here we demonstrate this procedure for the DNA aptamer MN4 in the presence of the thermolabile ligand cocaine, which rapidly converts to benzoylecgonine at high temperatures (&#62;60 &#176;C). Quinine is used as a negative control for ligand thermolability since it does not undergo conversion at these experimental temperatures and also binds to MN4. We describe the acquisition of thermolabile ligand DSC datasets and their analysis yielding thermodynamic and kinetic parameters of the folding, binding, and ligand conversion processes.

<table><tbody><tr><td>Sodium chloride</td><td>Chem Impex</td><td>#00829</td><td></td></tr><tr><td>Sodium phosphate monobasic dihydrate</td><td>Sigma Aldrich</td><td>71502</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S9763</td><td></td></tr><tr><td>Deioinized water for molecular biology</td><td>Millipore</td><td>H20MB1001</td><td></td></tr><tr><td>0.2 micron sterile syringe filters</td><td>VWR</td><td>CA28145-477</td><td></td></tr><tr><td>3 kDa centrifugal filters</td><td>Millipore</td><td>UFC900324</td><td></td></tr><tr><td>Dialysis tubing 0.5-1.0 kDa cutoff</td><td>Spectrum Laboratories</td><td>131048</td><td></td></tr><tr><td>Silicon tubing</td><td>VWR</td><td>89068-474</td><td></td></tr><tr><td>Plastic DSC flange caps</td><td>TA Instruments</td><td>6111</td><td></td></tr><tr><td>DNA aptamer MN4</td><td>Integrated DNA Technologies</td><td>https://www.idtdna.com/site/order/menu</td><td></td></tr><tr><td>Cocaine</td><td>Sigma Aldrich</td><td>C008</td><td></td></tr><tr><td>Quinine</td><td>Sigma Aldrich</td><td>22620</td><td></td></tr><tr><td>NanoDSC-III microcalorimeter</td><td>TA Instruments</td><td>http://www.tainstruments.com/nanodsc/</td><td></td></tr><tr><td>DSCRun software</td><td>TA Instruments</td><td>http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/</td><td></td></tr><tr><td>NanoAnalyze software</td><td>TA Instruments</td><td>http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/</td><td></td></tr><tr><td>Contrad-70</td><td>VWR</td><td>89233-152</td><td></td></tr></tbody></table>

measuring biomolecular dsc profiles with thermolabile ligands to rapidly characterize folding and binding interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

Representative data for the thermolabile ligand DSC are shown in Figure 1. The position and height of the thermolabile ligand-bound peak successively shifts down towards that of the unbound biomolecule as the thermolabile ligand is depleted with each scan (Figure 1a). The free denaturation profile is used as a reference for the endpoint of thermolabile ligand conversion (Figure 1b). Data for MN4 boun...

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Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

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Biochemistry

8.2K Views.  McGill University. Presenteren we een protocol voor snelle karakterisering van biomoleculaire vouwen en inbinden interacties met thermolabile liganden met behulp van differentiële scanning calorimetrie.

Video: Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

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 ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

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 ...

Biology

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

Degas the buffer, biomolecule, and ligand solutions in a table top degasser prior to loading into the differential scanning calorimeter, or DSC. Unscrew the pressure handle from the DSC. Then, run silicone tubing from the working buffer, and attach it to the front flange of the reference capillary.
Create a bridge between the reference and sample capillaries by connecting the rear reference flange to the front sample flange. Next, attach a piece of silicone tubing to the rear sample flange that runs to a waste flask with a vacuum line attached. Turn on the vacuum line to flush the DSC with 200 milliliters of working buffer.
Begin by attaching roughly 3- to 5-centimeter sections of silicone tubing to the reference capillary flanges. Next, insert a 1-milliliter pipette tip into the silicone tubing of the rear flange. Draw 0.8 milliliters of working buffer with a pipette, and insert the pipette tip with buffer into the silicone tubing of the front reference flange.
Gently press the pipette plunger down to pass the working buffer through the front silicone tubing into the reference capillary and up into the attached pipette tip of the rear flange. Press the pipette plunger down until the working buffer level reaches just above the front silicone tubing. Then, release the pipette plunger until the working buffer level reaches just above the rear silicone tubing. Continue passing the working buffer back and forth in the reference capillary to purge the volume of bubbles.
Next, cap the rear pipette tip with a forefinger and gently pull up on the rear pipette tip and front pipette to remove them from the reference flanges with the silicone tubing attached. Load the sample capillary with working buffer as before.
Place a black plastic cap on the rear reference and sample flanges, leaving the front flanges uncovered. Attach the pressure handle to the DSC and then, open the DSC software and pressurize the instrument by clicking the red &#34;Up&#34; arrow at the top of the interface once the power reading has stabilized. The DSC power is indicated in a box at the top right of the interface, along with the instrument temperature and pressure reading.
Equilibrate the DSC with working buffer by performing a forward and reverse scan. In the &#34;Experimental Method&#34; tab on the left side of the screen, ensure that the &#34;Scanning&#34; option is selected to run the DSC in temperature-scanning mode.
In the &#34;Temperature parameters&#34; inset under the &#34;Experimental Method&#34; tab, click the button for heating. Enter 1 and 100 degrees Celsius for the lower and upper experimental temperatures, 1 degree Celsius per minute for the scan rate, and 60 seconds for the equilibration period. Click the &#34;Add Series&#34; button under the input field for the equilibration period. Enter 2 into the &#34;Steps to add&#34; field in the pop-up window and check the &#34;Alternate Heating/Cooling&#34; box. Click &#34;OK.&#34;
The added scans appear in the lower portion of the interface. Check that the parameters for each scan are as desired. Start the experiment by clicking the green &#34;Play&#34; button at the top of the interface. Navigate to the desired window and input a file name for saving the experiment in the pop-up window. View the experiment progress by clicking the &#34;Data&#34; tab to the right of the &#34;Experiment Method&#34; tab.
Run reference experiments for baseline subtraction of the sample data by reloading the DSC with working buffer in both capillaries. Collect multiple forward and reverse scans over a suitable temperature range at 1 degree Celsius per minute with an upper equilibration time of 120 seconds.
Delete the previous buffer equilibration scans from the lower portion of the interface by highlighting each individually and clicking the red &#34;X&#34; to the middle right of the interface.
Add the new scans by clicking the &#34;Add Series&#34; button, entering 20 in the field for &#34;Steps to add&#34; and checking the &#34;Alternate Heating/Cooling&#34; box. Then, click &#34;OK&#34; and run the experiment by clicking the green &#34;Play&#34; button as before.
Repeat these steps with working buffer containing the desired concentration of ligand in both capillaries to obtain the reference experiments for the ligand. Collect two separate experiments to be used in acquiring the rate constant for thermolabile ligand conversion.
For the free biomolecule data set, ensured that the reference capillary contains the working buffer, while the sample capillary contains the free biomolecule at the desired concentration in working buffer. Next, run the sample experiments using the same DSC-loading procedure and experimental parameters used in the reference scans.
For the ligand-bound experiments, ensure that the ligand is in the working buffer in the reference capillary and the biomolecule plus ligand are in the working buffer in the sample capillary. Flush the system between additions of different ligands as before.
Perform one additional experiment with the biomolecule bound to the thermolabile ligand where the high-temperature equilibration period is increased to 600 seconds, and all other experimental parameters are the same as before. Proceed to data processing and analysis as described in the text protocol.

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

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