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Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions, as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studying RNA-protein interactions using ITC, and gives specific examples for monitoring the binding of Caenorhabditis elegans GLD-1 STAR domain to TGE RNA and the binding of Aquifex aeolicus S6:S18 ribosomal protein heterodimer to an S15-rRNA complex.
There are many studies in which ITC has been used to monitor protein-ligand, protein-protein, and protein-nucleic acid interactions(1–3). ITC measures the heat (q) evolved or consumed as aliquots of one reagent are added to a second reagent in a calorimetric cell. The reaction heat as a function of concentration is analyzed to obtain the complete thermodynamic characterization (ΔH, ΔG, ΔS) of the binding reaction (4). A full titration experiment can usually be completed in 2 hours.
As shown in Figure 1, within an adiabatic chamber the calorimeter contains a measurement and a reference cell(5). The measurement cell is filled with a solution of a macromolecule in the buffer of choice. The reference cell is filled with either water or the same buffer in which the macromolecule is dissolved. The tip of a syringe containing the ligand (either a small molecule or another macromolecule) is inserted in the measurement cell. Aliquots of the ligand are added to the measurement cell. The instrument maintains a small constant temperature difference between the measurement and reference cells. In response to binding of ligand to macromolecule in the measurement cell, which produces or consumes heat, the feedback system compensates to maintain the constant temperature difference between the measurement and sample cells. The signal measured during the titration is the rate of heating as a function of time, with a pulse corresponding to the addition of aliquots of ligand to the measurement cell. Integration of these pulses as a function of time yields a plot of q as a function of injection number, or molar ratio of ligand to macromolecule.
There is a great range in the size and complexity of RNA-protein complexes. These span simplest case involving a single protein binding to an isolated fragment of RNA to the large ribonucleoprotein complexes which involve multiple proteins binding to one or more RNAs.
The great variability observed in RNA-protein interactions makes ITC an ideal choice to study these interactions. In contrast to most other methods of detecting binding of RNA and proteins, there is no inherent size limitations to the macromolecules under investigation. Fluorescence anisotropy (6,7) requires that one of the two interacting components be much smaller than the other and surface plasmon resonance (8) requires that one of the two components be immobilized. Gel mobility shift assays require that the migration of an RNP complex in the gel is sufficiently different than the free RNA, which is less likely when complexes between small proteins and large, highly structured RNAs are studied. Gel shift assays have the requirement that the RNP complex is kinetically stable during electrophoresis, which is often not the case for weak binding complexes. ITC does not require labeling or immobilization of either macromolecule, is performed under equilibrium conditions, and can be performed under a wide variety of buffer conditions.
The amount of material required is an important factor to consider when performing an ITC experiment. Unlike gel mobility shift assays or fluorescence anisotropy measurements, in which the labeled molecule is often present in trace quantities, ITC requires nanomole quantities of each reactant. To obtain a reliable value for the association constant of an interaction, the appropriate concentrations of reactants must be used. As described by Wiseman et al (5), the parameter c, which equals the association constant (Ka) times the total concentration of the reactant in the calorimeter cell (Mtot), should lie between 1 and 1000. A more reliable estimation of Ka can be obtained if the value of c is kept between 10 and 500. A sufficient concentration of titrant should be used so that its concentration at the completion of the titration will be 1.5–2 × n (the number of binding sites per molecule) times the concentration of reactant in the cell. For a titration in which a total of 250 μl of protein is added to a reaction cell containing approximately 1.5 ml of RNA, the protein should be 10–20n times the concentration of RNA.
Two different systems are chosen to demonstrate both a simple protein-RNA interaction and a protein-RNP interaction.
Below is a step-by-step protocol for running an ITC experiment to study the binding of a single protein to RNA. To study the binding of a protein to an RNP complex, the only modification required is the formation of the RNP complex prior to degassing the samples (Section 3.1.4, following step 1, see note 4)
M.I.R. was supported by Research Scholar Grant #PF-01-087-01-GMC from the American Cancer Society. S.P.R. is supported by the Damon Runyon Cancer Research Foundation Fellowship (DRG-1723). This research was supported by grants from The Skaggs Institute for Chemical Biology and the National Institutes of Health (NIH, GM53320 and GM53757).
1Only 500 μl of this sample will be used for the experiment and control titrations, but additional protein is needed so that the injection syringe can be filled without introducing bubbles. The needle contains two holes, one at the tip and another on the side. The hole on the side must be covered by protein sample during filling, necessitating the additional volume.
2Depending on the buffer in which the proteins are stored, it may be necessary to prepare an additional 4 liters of dialysis buffer and change the buffer approximately 8 hours into the dialysis. This is particularly important if the protein is stored in a salt concentration that is much higher than in the measurement buffer or if a large volume of protein (>10 ml) is being dialyzed.
3The concentration of the proteins used in this example was determined using the calculated extinction coefficient at 280 nm(14). Other methods may be used to determine concentrations of proteins, but inaccurate concentration determination will effect all parameters in the subsequent determination of ΔH, ΔG and n.
4The RNA samples should be annealed prior to degassing, as the heating process will liberate more dissolved air. If an Aquifex aeolicus S15-RNA complex is to be titrated, form the complex before degassing by incubating the annealed RNA with an equimolar amount of protein for 5 minutes at 65 °C. The Aquifex aeolicus protein is stable under these conditions.
5It is possible, though not recommended, to perform the titration with protein in the measurement cell and RNA in the syringe. The initial injections will create a situation in which protein is in great excess over RNA, which can produce complexes that are prone to aggregation/precipitation.
6The injection parameters will vary based on the specific interaction being measured. The values given have yielded good results with both systems described here. The first injection is kept small because there is the possibility of some mixing of protein solution on the outside or in the tip of the needle during the equilibration process, resulting in an erroneous data point. The heat evolved during the first injection will not be included in the data fitting, but the amount of protein in this injection will be used to calculate the total ligand concentration in the reaction cell.
7A control titration such as that described is not always necessary. If there is only a single binding site for the protein on the RNA, and it is fully saturated by the end of the titration, the heat observed following saturation can be used as the dilution reference.
8For particularly high affinity interactions (Ka ≥ 1 × 109), the value of Ka may need to be held constant as it will not be well defined unless the value of c can be kept between 10 and 500. If a competitive inhibitor binds to the same site as a high-affinity binding ligand, one can determine the Ka of the high-affinity ligand using displacement isothermal titration calorimetry (15).