Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler
Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler
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DOI：
10.2144/0000113922
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发表时间：
2012-10-01
期刊：
影响因子：
2.7
通讯作者：
Storey, Kenneth B.
中科院分区：
文献类型：
--
作者：
Biggar, Kyle K.;Dawson, Neal J.;Storey, Kenneth B.
蛋白质稳定性可以通过许多不同的技术来监测。然而，这些方案通常是冗长的，消耗大量的蛋白质，并需要昂贵的和专门的仪器。在这里，我们提出了一个新的协议，使用定量实时热循环仪分析蛋白质去折叠动力学。该技术能够在多孔平台中分析各种变性剂（及其与温度变化的相互作用）对蛋白质稳定性的影响，其中样品可以在几乎相同的条件下并行运行，并具有高灵敏度检测。使用这种设置，研究人员可以评估蛋白质变性的半最大速率（K-nd），最大变性速率（D-max）以及蛋白质解折叠中单个变性剂的协同性（μ系数）。溶菌酶和己糖激酶被用作模型蛋白质和尿素作为模型变性剂，以说明这种新的方法和它提供的蛋白质解折叠的动力学。总的来说，这种方法允许研究人员在同一测定中在恒定或可变温度下探索大量变性剂，提供以前无法获得的变性动力学估计。
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 (mu-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.