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Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler
 
Kyle K. Biggar*, Neal J. Dawson*, and Kenneth B. Storey
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When comparing the experimentally derived Knd (2.69 ± 0.06 M urea) to literature values of C1/2 (5.69 ± 0.14 M urea; where C1/2 is the urea concentration at which protein is 50% unfolded) for HEWL in urea, the experimentally derived Knd is significantly different than the experimentally determined C1/2 value (17). Although these results are measurements of different unfolding characteristics of HEWL (50% rate versus 50% amount unfolded protein), these results together may suggest that the rate of protein unfolding reaches the fastest possible rate of HEWL unfolding before the protein is completely unfolded. Interestingly, previous studies suggest that HEWL retains its overall structural conformation at urea concentrations below 2 M urea (17). However, these studies determined relative protein unfolding by tryptophan fluorescence at 360 nm and the initial periods of unfolding may be below the limit of detection. In the case of native protein denaturation, hydrophobic residues that are exposed can interact and be detected with the SYPRO Orange dye. This may allow for a lower limit of detection, where the initial stages of protein unfolding can be detected, providing a better representation of the unfolding process. Native protein denaturation allows researchers to monitor the rate of unfolding in real-time during the entire progression, providing a greater resolution of the unfolding process.

Here we have presented a new technique to quantify the kinetics of protein denaturation. We obtained a half-maximal rate of native protein denaturation (Knd), maximum rate of denaturation (Dmax), and extent of denaturant cooperativity (µ-coefficient) under several experimental conditions. Our method is very simple, requires only small sample volumes, and provides large amounts of reproducible real-time data in a relatively short experimental protocol. Finally, this method will allow the researcher to quickly and efficiently determine the susceptibility of protein structure to many different denaturants while also allowing the variable of temperature change to be used as a means to further manipulate denaturation. Stability data could also be collected as a function of competing stabilizing agents. However, this study may be limited by the rate of SYPRO incorporation relative to extremely high rates of protein unfolding. Additionally, proteins must contain sufficient hydrophobic regions to interact with the SYPRO Orange dye; a property that will influence the sensitivity of the technique for each particular protein of interest. This method provides an opportunity for researchers to explore protein stability with readily available equipment, and allows current studies that evaluate protein stability to explore changes in degradation kinetics.

Acknowledgments

Thanks to J.M. Storey for an editorial review of the manuscript. This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada. K.B.S. holds the Canada Research Chair in Molecular Physiology, K.K.B. held an NSERC postgraduate fellowship.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to Kenneth B. Storey, Institute of Biochemistry & Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada. Email: [email protected]


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