title: Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler.
abstract: 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 (K(nd)), maximum rate of denaturation (D(max)), 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.
authors.affiliation: Institute of Biochemistry & Department of Biology, Carleton University, Ottawa, Ontario, Canada.
authors.forename: Kyle K
authors.initials: KK
authors.lastname: Biggar
authors.forename: Neal J
authors.initials: NJ
authors.lastname: Dawson
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Storey
mesh_terms: D004055:Differential Thermal Analysis; D007700:Kinetics; D011489:Protein Denaturation; D055550:Protein Stability