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

Biggar, K. K., Dawson, N. J. and Storey, K. B. (2012) Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler. BioTechniques, 53(4), pp. 231-238. (doi: 10.2144/0000113922) (PMID:23046506)

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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 (Knd), maximum rate of denaturation (Dmax), 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Dawson, Dr Neal
Authors: Biggar, K. K., Dawson, N. J., and Storey, K. B.
College/School:College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
Journal Name:BioTechniques
Publisher:Future Medicine Ltd.
ISSN:1940-9818
ISSN (Online):1940-9818

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