Dsc Itc Modern Methods Examining Protein Protein Interactions Biology Essay

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Living things requires a continuous throughput of energy. Bioenergetics describes how the organism gain, channel and use this energy. Nearly all biological processes involve protein-protein or protein-ligand interactions that drive the formation and disassembly of intracellular signaling pathways with the release or absorption of heat. If unregulated, this will result in diseased condition. The structural information obtained through x-ray crystallography and NMR spectroscopy shed no light into the energetic forces that drives the biological processes responsible for maintaining the folded conformations of biomacromolecules in solution or receptor-ligand interactions. To understand the role of a biomacromolecule and modulating its function to develop novel therapeutic compounds, it is necessary to understand the thermodynamic and kinetic aspects of the underlying biophysical process. Calorimeter is a device that is used to measure the heat absorbed or evolved during a chemical reaction or a biophysical change.

Advances in the field of genomics and proteomics, has opened new vistas for biocalorimetry to be used in drug discovery to monitor real time interactions of biological significance and to find out the nature of energetic forces responsible for driving the protein folding or its interaction with small molecules. Ref 5-7, chapter 3. With the help of this, we can predict effect of mutations as well as design specific inhibitors as drugs.

There are several methods available for measuring heat changes in the biochemical systems. The two most common techniques are Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC). DSC is used to monitor changes associated with phase transitions whereas ITC to monitor the energies of interaction between molecules upon mixing.

Book: New design strategies for ligands that target protein-protein interactions, D.S. Lawrence, Springer-Verlag, 2005.

Instrumentation

DSC continuously measures the apparent specific heat of a system as a function of temperature. Therefore it can be used to measure a heat induced phase transition or conformational change. In this two identical cells are placed in nearly adiabatic condition and the temperature of the instrument should be kept atleast 5-10oC below than that at which the experiment is to be carried out.

Figure:

Energy is introduced simultaneously into a sample cell (containing the aqueous buffered solution of a protein of interest) and reference cell (having only the identical buffer) raising the temperature of both in the range 0.1-100oC identically over time. The difference in the input energy required to match the temperature of the sample to that of the reference at each step of the scan is the amount of excess heat absorbed (in endothermic process) or released (in exothermic process) by the macromolecule.

Here, we start the experiment at a temperature where the protein is in its nativefolded state and determine its heat (CN). As the temperature is increased, the protein will undergo thermal unfolding and subsequent slope will change and represent the heat capacity of the denatured protein (CD). The change in enthalpy associated with denaturation can be determined by integrating the area under the curve between T1 and T2, where T1 is the temperature at which all of the protein is in its native conformation and T2 where it is denatured. (Figure). The temperature at which half the protein is denatured is the melting temperature (Tm) for the protein. This is the point at which the amount of denatured equals the native form of the protein.

Although DSC can be used to determine the binding energetics of a reaction, the method to study the enthalpy change for a biomolecular binding interaction resulting from mixing together two or more components is by ITC. This interaction produces heat effects which may be due to heat of interaction, dilution of ligand or macromolecule, stirring etc.

Biological systems that can be studied by ITC are Protein-protein, protein-ligand interactions involved in processes, such as cell signaling, Alzheimer's disease, molecular chaperones, protein-drug, drug-DNA interactions etc.

Principle:

Melting curve:

dQ = Cp * n * dT

Cp = (1 /n) * (dQ/dt))(dt/dT)

= (1/nσ)(dQ/dt)

Where σ = scan rate = dT/dt

Differential power = dQ/dt

Cp is calculated from the added power and scan rate

Figure:from slides (melting curve)

Disadvantage: Any interaction that is a athermal will not be reported. (i.e. entropy dependent)

In this a sample of target protein is placed into the calorimeter, which is insulated from the outside world. It involves incremental addition over time of a ligand in a series of small injections into a solution of a protein. At each titration point, the interaction results in the release or absorption of heat (q). At the initial time intervals the amount of free protein is sufficient to bind ligand. With time the magnitude of heat change decreases as the sites on the target protein becomes saturated. Modern instruments actually measure the amount of electric current required to maintain a constant temperature between the sample cell and a reference over time and 'q' is integrated as a function of time. On reaching thermal equilibrium, a peak is obtained (Figure). Even after saturation of all the binding sites, heat is evolved; it is because of the heat of dilution of the interacting component being dissolved into the solution in the cell. In the figure, area under the peak represents the enthalpy change upon single injection. We integrate this to get ΔHo of the complete process

ITC cannot be directly used to study the thermodynamics of a binding reaction in which Ka is very large. In such tight binding reactions, each incremental addition of ligand results in complete binding of the ligand. The resulting titration will show a sharp transition from complete binding of the added ligand to no binding. One strategy to circunvent this problem is through a competitive ITC experiment, where a weak ligand initially bind to a protein is displaced is displaced by the one that binds the protein more tightly.

Principle:

A ligand [L] is introduced into a solution of protein [M] that can bind to form a simple binary complex [ML]. The resulting release or absorption of heat from the reaction is dependent on the enthalpy at a particular temperature and the number of moles of complex formed

Energetics of interaction between a macromolecule [M] and ligand [L] is represented by the equation: M + L ↔ ML

ITC measures the heat associated with the equilibrium and 'K' and from this, the value of ΔGo and ΔSo can be calculated by using the following formulae

With equilibrium constant, K = e-ΔGo/RT

Free energy can be determined by ΔGo = ΔHo - TΔSo

Q = ΔH * [ML] * V

where [ML] = concentration of the bound ligand

and V = sample volume

K = [ML] / [M][L] = [ML] / ([MT]-[ML])([LT]-[ML])

where [MT] = total macromolecule concentration

and [LT] = total ligand concentration

on solving

Q = ½ * ΔH * [MT] * V {1 + x + C - ((1 + x + C)2 - 4x) ½}

With x = [L] / [M] and C = 1 /(K[M])

Role of Biocalorimetry in Drug Discovey

To identify new chemical entities (NCEs) that have the ability to treat a particular disease state, there is a need to identify biological targets and to elucidate its 3D structure and biological function. With the help of genetic analysis we can identify the potential targets. After the initial identification of the biological target , we have to validate that whether that target is responsible for the particular diseased state. For this we require sufficient quantity of purified protein. ITC can be used to check the protein preparation to be used in bioassays, screening and structural analysis of proteins.

Following the identification of the target, the next stage is the development of highthroughput screening of the compounds (hit identification) for the desired activity against that particular target. The next stage is the lead identification in which large number of analogues of the compounds which displays the activity are synthesized. At this stage of drug discovery, there is a need to develop bioassays capable of measuring the protein-protein and protein-ligand interactions. Biocalorimetry can be used as one of the available technique to characterise these interactions.

After the identification of the lead compound, it is necessary to determine the affinity of that compound with the target receptor. For this, we can test the effect of a ligand on biological activity of the desired compound. On interaction of each protein with a particular ligand will show difference in their ITC data, which furthers helps in the identification of the target.

Further, we have to enhance its activity and selectivity towards a particular target. There is also a need to check its bioavailability as even highly potent compounds lack activity in vivo. This can be done by bioisosteric replacement in the original lead to produce newer molecules with improved biological properties. As the ligand can also ineract to proteins other than the receptor like plasma proteins, transport proteins etc. its biological activity is underestimated. ITC is very sensitive instrument to quantitate the strength of these interactions.

Various examples of protein-protein and protein-ligand interactions have been characterised by DSC and ITC as reported in literature. Kirkitadze et al have characterised independently melting modules and highly structured intermodular junctions within complement receptor type 1 (CR1) by DSC [Ref.]. It was shown that as temperature or denaturant was increased, the 15-16 junction appeared to melt first, followed by melting of both modules. Also, the 16-17 junction appeared to melt first (50.5oC), followed by the 15-16 junction, and module 17 itself (57.5oC); finally, modules 15 and 16 became denatured (61.5oC). Comparison of 1H-15N HSQC spectra for single, double and triple module fragments indicates that module 16 makes more extensive contacts with module 15 than with module 17.

Kirkitadze MD, Krych M, Uhrin D, Dryden DT, Smith BO, Cooper A, Wang, Hauhart R, Atkinson JP, Barlow PN.

Biochemistry, 1999 Jun 1;38(22):7019-31.

Also examples of antigen-antibody interaction that have been characterised by ITC are reported by various research groups.

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