Introduction

Protein thermal stability testing, also known as thermofluor (TF) or differential scanning fluorimetry (DSF) (Ericsson et al., 2006; Niesen et al., 2007) , is based on the measurement of the increase in the thermal stability of the protein induced by ligand binding. The approach consists of applying gradually increasing temperature on purified proteins, in the presence and absence of a ligand. The protein melting temperature is defined as (Tm), which is known as the

midpoint of the protein unfolding position. The Tm value (the inflection point of the transition

curve, figure 2.1), can be calculated using the following equation, derived from the Boltzman equation:

( )

63 where “LL” and “UL” are the values of minimum and maximum intensities, respectively, “a” denotes the slope of the curve within Tm , “y” and “x” denote the fluorescence intensity and the

temperature, respectively (Vedadi et al., 2006; Cummings et al., 2006; Niesen et al, 2007).

In the DSF approach an “environmentally sensitive” dye which specifically interacts with non- native protein is used as an indicator to observe the amount of unfolded protein in solution as a function of temperature (Mezzasalma et al., 2007; Cummings et al., 2006). This is achieved by measuring the fluorescence from the dye which results from the changes in the dye’s emission properties upon interaction with unfolded protein (Vedadi et al., 2006). DSF uses a number of fluorescent dyes which differ in their optical properties. The dyes’ fluorescence is quenched in an aqueous solution, whereas in non-polar environment, such as the hydrophobic sites on unfolded proteins, the fluorescence intensity becomes significantly higher. With a high signal to noise ratio, SYPRO orange is currently the dye with most favourable properties for this approach. It has a relatively high wavelength for excitation (near 500 nm) which reduces the chances of any small molecule causing its fluorescence intensity to quench and therefore interfering with its optical properties (Niesen et al., 2007). The emission wavelength of SYPRO orange is near 600nm (Niesen et al., 2007). Some proteins may show no unfolding transition when analyzed using SYPRO orange, in which case an alternative dye should be tested. The molecular structure of SYPRO orange is not disclosed, (symbolized as a three-ring aromatic molecule in figure 2.1) (Niesen et al., 2007).

Most ligands that bind specifically to the native protein will increase the Tm, and a temperature

shift (∆Tm) between the protein’s melting temperatures in the two conditions is observed and

measured. The extent of this shift is believed to be proportional to the ligand’s binding affinity for the protein (Cummings et al., 2006). Ligands that bind non-specifically are associated with

64 aggregation which destabilises the protein tertiary structure and do not test positively in DSF (Cummings et al., 2006).

SYPRO orange Protein

Exposure of hydrophobic parts as protein

unfolds

Gradual removal of protein from solution

due to precipitation and aggregation

Strong fluorescent light of 610 nm emitted by the dye molecules bound to non-

polar parts

Excitation of a basic fluorescence intensity

by light of 492 nm

Figure 2.1: fluorescence intensity plotted as a function of temperature for the unfolding of

protein (citrate synthase). The plot has a sigmoidal shape which is illustrated by a two-state transition depicted in this figure. The lower level (LL) and upper lever (UL) of the fluorescence intensity defined by equation 2.1 are also demonstrated here (figure taken and modified from Niesen et al, 2007).

A key advantage of the DSF approach is its applicability to a wide range of proteins. In addition to this, it is a rapid and cost effective approach that requires relatively small amounts of protein and can be implemented in any laboratory (Vedadi et al., 2006). Furthermore, unlike other screening methods such as NMR, DSF sets no upper limit on the binding affinity that can

65 be measured. It is possible to screen for compounds with expected affinity (KD) between 1 nM

and 1 mM. However, varying protein and compound concentrations is frequently necessary. For example, higher concentration may be required for compounds that bind with relatively low affinity, such as nucleotides. Also, it can be sometimes necessary to increase protein concentration to obtain an acceptable signal-to-noise ratio (Niesen et al, 2007).

It also provides additional information on the ligand’s binding mechanism, making it possible to distinguish protein stabilizers from protein destabiliseers. Another main advantage of the approach is that it is applicable to different stages of the screening process, from primary screening to hit profiling. It is also a useful selection tool of protein constructs for use in screening and X-ray crystallography (Cummings et al., 2006). DSF also plays an important role in the expression and purification of proteins since it allows the detection of a specific ligand that improves the protein stability and hence decreases its potency to unfold and reduces the chances of its aggregation and susceptibility to proteolysis (Vedadi et al., 2006).

The DSF approach has limitations that are known to make it difficult to calculate the Tm value.

This may arise from a number of proteins not being folded in their native state and, therefore, have no hydrophobic core. On the other hand, some proteins can be thermo-resistant or have a thermal stability higher than the temperature range applied. Other proteins may have a hydrophobic core partly exposed in their native state. This results in a high initial fluorescence due to the dye interacting with the non-polar parts exposed. Only a small transitional increase in fluorescence may be observed due to the dye directly interacting with the compounds tested or ingredients in the solvents used. Finally, some transition curves observed are not monophasic which is due to the protein comprising more than one domain or forms oligomers (Niesen et al., 2007).

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