To determine the effects of urea concentration on protein unfolding rates, protein samples were incubated at various urea levels in the presence of SYPRO Orange dye. At each urea concentration, the relative rate of unfolding of the protein was recorded as an increase in the fluorescence of SYPRO Orange over time. The changes in fluorescent values were then plotted as a function of incubation time. Data points were truncated to display only the initial linear portion of the denaturation curve (Figure 2A and 3A). The slope of this relationship was then calculated for each urea concentration using a linear regression of fluorescence with respect to incubation time. Slopes increased with increasing urea concentration. Importantly, it should be noted that this technique will not be able to measure the initial rates of rapidly unfolding proteins as there is a dead time of ~5 s between the addition of protein and the initial fluorescent reads from the thermocycler. To determine the rate of HEWL and HK denaturation in urea, each resulting slope (calculated above) was then plotted against the concentration of urea. These values gave a sigmoidal plot that was analyzed using the Hill equation (Figure 2B and 3B) (12). Interestingly, we have found that our plots of unfolding rates for HEWL are similar to those by Laurents and Baldwin, whose study used tryptophan fluorescence; however, comparable rates of unfolding could not be obtained (13).
The concentration of urea that results in half-maximal rate of protein denaturation is defined as the kinetic constant of native denaturation (Knd). The Knd at 25°C was determined to be 2.69 ± 0.06 M (n = 4) for HEWL and 3.31 ± 0.22 M (n = 4) for HK. As urea concentration increases, protein will denature at a faster rate until a maximum rate is reached. This maximum rate, Dmax, can be calculated from an asymptote determined from the Hill equation. This Dmax value, therefore, represents the upper limit of the rate of protein denaturation (the change in fluorescent units per minute or ΔF.U./min). From the present data, a Dmax of 1636 ± 147 ΔF.U./min was calculated for HEWL and 124 ± 8 ΔF.U./min was calculated for HK denaturation by urea at 25°C.
The extent of cooperativity of the denaturant can also be uncovered when calculating the apparent Hill coefficient. As urea molecules interact with a protein there is a disruption of intra-molecular interactions. Upon the disruption of these intra-molecular forces, the protein becomes increasingly accessible to subsequent urea molecules. This results in a cooperative effect on protein unfolding (14). Various denaturants interact with protein in a variety of distinct mechanisms. As a result, the cooperativity of these interactions are critical to understanding the complete effect of the denaturant on each protein of interest. This is especially true at lower concentrations of denaturant, where the degree of cooperativity yields a large effect on the rate of denaturation of the protein. This measure of cooperativity, defined herein as the µ-coefficient, for the influence of urea on protein denaturation was calculated to be 2.55 ± 0.17 for HEWL and 5.65 ± 0.62 for HK. These results suggest that urea has a more pronounced effect on the rate of HEWL unfolding compared with HK.
The rate of unfolding for a given protein can be influenced by the choice of chemical denaturant as well as by environmental factors such as temperature and pH. One important advantage of our thermocycler-based method is the ability to utilize the thermal block for very precise control over sample temperature as well as the capacity to look for interacting effects of chemical denaturant and temperature. Many gradient-enabled thermocyclers can incubate and monitor samples at a wide range of temperatures in a single assay. For example, for a given experiment, the BioRad MyIQ2 thermal block can accommodate a temperature range of 25°C within the limits of 4°C and 99°C. Using this ability to monitor fluorescent changes at multiple temperatures, we assessed the influence of temperature on the Knd of urea for HEWL and HK in 10°C increments in separate trials over the range of 5°C to 35°C (Figure 2C and 3C). No significant influence of temperature on Knd was seen between 5°C and 35°C (P < 0.05) for HEWL and no significant influence of temperature on Knd was seen between 5°C and 15°C (P < 0.05) for HK. These regions may represent temperature ranges in whichKnd is independent of an increase in temperature. Indeed, previous studies have shown that lysozyme is thermostable up to ~40°C and is 50% denatured at ~50°C (15). However, at higher temperatures, 25°C and 35°C, the Knd for urea of HK significantly decreased and suggests a combined influence of denaturant (urea) and temperature on protein unfolding. The effect was pronounced, with Knd dropping from ~5 M at the lower temperatures to ~1 M at 35°C, demonstrating that urea is a much more effective denaturant at higher temperatures. Perhaps this is because the native conformation of the protein at 35°C would be expected to be freer because of the higher temperature on protein and/or differential effects of high temperature on the hydrophobic versus hydrophilic bonds that underlie protein folding and conformation (16).