*K.K.B. and N.J.D. contributed equally to this article
Protein stability can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. Here we present a new protocol to analyze protein unfolding kinetics using a quantified real-time thermocycler. This technique enables the analysis of a wide range of denaturants (and their interactions with temperature change) on protein stability in a multi-well platform, where samples can be run in parallel under virtually identical conditions and with highly sensitive detection. Using this set-up, researchers can evaluate the half-maximal rate of protein denaturation (Knd), maximum rate of denaturation (Dmax), and the cooperativity of individual denaturants in protein unfolding (µ-coefficient). Both lysozyme and hexokinase are used as model proteins and urea as a model denaturant to illustrate this new method and the kinetics of protein unfolding that it provides. Overall, this method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay, providing estimates of denaturation kinetics that have been previously inaccessible.
The limits of survival for many living organisms are marked by extreme environmental conditions: for example, 0 to 12.5 for pH and approximately −18°C to 121°C for temperature (1). While some organisms that live in extreme environments can evade the stress or employ defensive mechanisms that allow them to sustain favorable intracellular conditions, other organisms must adapt their intracellular molecules for survival. For example, the intracellular pH of many acidophiles, neutrophiles and alkaliphiles are regularly maintained at around 6.5, 7.5, and 9.5, respectively (2). To maintain proper functioning of their intracellular proteins, these organisms display structural adaptations that influence both protein stability and activity (3). Apart from the ability of some species to exist at high and low intracellular pH, many organisms have the ability to tolerate extreme temperatures. For example, many organisms have successfully colonized cold environments
and have evolved mechanisms to overcome the life-endangering consequences of low temperature with structural and functional adaptations of their macromolecules that include adaptations of protein structure, stability and post-translational modifications (4). Likely candidates for adaptive changes are those proteins with crucial functions necessary for survival (5-7). Consequently, there is great interest in understanding the mechanisms involved in stabilizing protein structure, not only for extremophiles, but for modified protein structures in general.
It has been known for many years that proteins can be unfolded in aqueous solution by high concentrations of denaturants, such as guanidine hydrochloride (GnHCl) or urea. Denaturation using these chemicals are one of the primary methods of determining protein structure, comparing the stability of modified to native proteins. Denaturants alter the equilibrium between the native (folded) and denatured (unfolded) states of the protein:
where N is the concentration of protein in the folded or native conformation, and D is the concentration of protein in the unfolded or denatured state at a particular denaturant concentration. The relative concentrations of N and D are typically determined with the use of either (i) fluorescent probes to monitor the conformational state of the protein or (ii) proteolysis combined with SDS-PAGE (known as pulse proteolysis) to monitor relative amounts of denatured (unfolded) or non-denatured (folded) protein. Although this type of experiment can provide information about the free energy of protein stability, it does not provide information regarding the rate of protein denaturation. Determining the kinetics of native protein denaturation could provide the researcher with detailed information regarding proteinstructure and the influence of various effectors on protein stability and unfolding rate.
The determination of protein stability is an essential step in characterizing protein structure. There are many traditional techniques, such as fluorescence, circular dichroism spectroscopy, hydrogen exchange-mass spectroscopy, differential scanning fluorometry (DSF), protein crystallization, and pulse proteolysis used to determine the structural stability of proteins and enzymes. These methods determine stability by monitoring conformational changes induced by either thermal or chemical denaturation. However, with the exception of DSF, these methods require substantial amounts of pure protein and expensive specialized instrumentation. Unfortunately, although DSF is able to utilize low amounts of protein, the technique only monitors the thermal stability of protein structure as a sample is exposed to increasing temperature (8). In the present study, we expanded the method of DSF to evaluate denaturation kinetics of proteins when exposed to a denaturant at either constant or varying temperatures.
Existing DSF methods work by utilizing a modified qRT-PCR thermocycler to monitor thermal unfolding of a protein in the presence of a hydrophobic fluorescent dye (typically SYPRO Orange). As a result, the use of DSF is applicable to a wide range of proteins that contain hydrophobic regions within the protein structure. The presence of a hydrophobic region is critical, since the SYPRO Orange dye used for DSF is highly fluorescent upon binding to hydrophobic sites on unfolded proteins. The typical analysis conducted using DSF is to determine the change in fluorescence intensity as a function of increasing temperature; this typically generates a sigmoidal curve that can be described by a two-state transition (8). The relatively high wavelength for excitation of SYPRO Orange (488 nm) also decreases the likelihood that small molecules would interfere with the optical properties of the dye. In the present study, we present important modifications to the DSF procedure to obtain real-time kinetic analysis of protein unfolding. Our modifications expand the DSF technique by altering the experimental protocol, allowing the researcher to obtain real-time fluorescent data over a constant (or gradient) temperature and collect data at regular intervals (between 1 s and 99 min 59 s). By combining the use of (i) a 96-well PCR microplate, (ii) relatively small sample volumes (20 µL), (iii) a short procedure time, and (iv) the ability to monitor denaturation at various temperatures (typically ranging from 4.0°C – 99.0°C on a gradient-enabled qRT-PCR thermocycler), this protocol allows researchers to combine multiple experimental conditions into a single experiment, reducing both time and technical variation. This method is a highly accurate and reproducible way to assess protein stability and determine the rate of protein denaturation under environmental conditions including variable pH, denaturants (e.g urea and GnHCl) and stabilizers (e.g glycerol and trehalose).