As a result of a broad range of excitation and emission fluorescence, SYPRO Orange (Invitrogen, Burlington, ON, Canada, Cat. No. S-6650) can be monitored in quantitative RT-PCR instruments using the filters commonly provided with the machines: FAM (485 nm) and ROX (625 nm) for excitation and emission, respectively (Figure 1). For the presented set of experiments, we modified a BioRad MyIQ2 thermocycler (BioRad, Hercules, CA, USA, Cat. No.170–9790), installing the appropriate filters into a separate channel than the one utilized for SYBR-green based experiments. This allowed continued functionality for both qRT-PCR and protein denaturation experiments. A pure dye calibration was carried out for SYPRO Orange. No further modifications were carried out to the instrument.
Protein denaturation kinetics
Lysozyme purified from hen egg whites (HEWL) (Worthington Biochemical Corp., Lakewood, NJ, USA, Cat. No. LS002931) was diluted to a concentration of 25 mg/mL in potassium phosphate buffer (100 mM potassium phosphate, pH 7.0, 150 mM NaCl), and hexokinase (HK) purified from Saccharomyces cerevisiae (Sigma Aldrich, St. Louis, MO, USA, Cat. No. H-5500) was diluted to a concentration of 0.2 mg/mL in potassium phosphate buffer. Optimal protein concentrations were determined using the method outlined below and were found to provide a suitable dose-response relationship when compared with other protein concentrations. This buffer was previously shown to be suitable for studies of protein denaturing conditions and is commonly used in experiments involving SYPRO Orange (8). The SYPRO Orange dye was diluted to a 40× stock (based on the 5000× stock solution as supplied by Invitrogen) in potassium phosphate buffer and used from this concentration for all experiments.
All experiments using the qRT-PCR thermocycler were carried out in 96-well thin-walled unskirted PCR microplates (BioRad, Cat. No. MLP-9601). All incubations with urea were conducted at 11 different final concentrations ranging from 0 to 4.5 M in potassium phosphate buffer. Each sample consisted of 12.5 µL of potassium phosphate buffer containing various concentration of urea, 5 µL of purified protein and 2.5 µL of 40× SYPRO Orange dye for a final volume of 20 µL. Fluorescent measurements were initiated ~5 s after the addition of protein to the samples. Fluorescent reads were taken every 5 s at 5°C, 15°C, 25°C, and 35°C for 50 min, using the manufacturer-supplied software (version 3.0.6070). Temperature incubations were carried out in separate experiments. Data were analyzed using SigmaPlot v.11 software (Systat Software, Chicago, IL, USA). The change in fluorescence was plotted using a simple scatter plot. The whole unfolding curve was measured in the experiment and fluorescence was found to rapidly increase initially andthen reach a plateau (Supplementary Figure S1). The initial unfolding rates of the curve were truncated and analyzed using a linear regression of fluorescence at various concentrations of urea. All experiments were carried out at a concentration of SYPRO Orange within a linear region of fluorescence that displayed no significant changes in protein unfolding rate. The concentration of SYPRO used in the study is comparable to other protein unfolding studies (8).
The resulting slopes were plotted on a second graph using the Kinetics v 3.5.1 program and analyzed using the Hill Equation (9). From this, Knd, Dmax, and µ-coefficient values were calculated. Respectively, these values were defined as the concentration of denaturant (urea in this case) that resulted in a half-maximal rate of native protein denaturation (Knd), the calculated maximum rate of protein denaturation (Dmax), and the extent of denaturant cooperativity (µ-coefficient) calculated in a similar manner to that of a Hill coefficient. Data are expressed as mean ± sem from multiple trials. Statistical testing used Student's t-test.Results and discussion
Studies characterizing protein unfolding can help researchers to understand general questions of protein stability under various conditions. The methodology described in this study extends the range of options for researchers interested in investigating protein stability. By using modified qRT-PCR equipment, it is possible to analyze the effects of a wide range of denaturants (and their interactions with temperature change) on protein stability in a microplate platform where multiple 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 protein denaturation (Dmax), and the cooperativity of individual denaturants in protein unfolding (µ-coefficient). As a protein unfolds, it exposes hydrophobic residues that are able to interact with SYPRO Orange dye, resulting in an increase in fluorescent emission that can be detected by qRT-PCR optics. In the present study we used two model proteins (lysozyme and hexokinase) and a model denaturant (urea) to demonstrate this new method and the kinetics of protein unfolding that it can supply. HEWL was chosen as a model protein as it is a well-studied monomer that has been shownto unfold in a relatively simplistic model of a two-state transition, while yeast hexokinase (HK) is a dimer also been shown to undergo a two-state transition of unfolding (10,–11).