P07 : Real-time protein unfolding: A method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler

Abstract

The stability of a protein can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. In this study, we present a new protocol to analyze the kinetics of protein unfolding utilizing a quantitative real-time thermocycler. 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 on 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 determine the concentration of denaturant that results in a half-maximal rate of protein denaturation (KND), the maximum rate of denaturation (Dmax), and the cooperativity of individual denaturants in protein unfolding (I1/4-coefficient). In the present study we use hexokinase as a model protein and urea as a model denaturant to illustrate this new met hod and the kinetics of protein unfolding that it can supply. This method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay. Overall, this method provides an estimate of denaturation kinetics that was previously inaccessible.