Revisiting absorbance at 230 nm as a protein unfolding probe
Revisiting absorbance at 230 nm as a protein unfolding probe
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DOI：
10.1016/j.ab.2009.03.028
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发表时间：
2009-06-15
影响因子：
2.9
通讯作者：
Park, Chiwook
中科院分区：
文献类型：
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作者：
Liu, Pei-Fen;Avramova, Larisa V.;Park, Chiwook
蛋白质的热力学稳定性和去折叠动力学通常通过用光谱探针（例如圆二色性（CD）和荧光）监测蛋白质去折叠来确定。还已知230 nm处的UV吸光度（A（230））对蛋白质构象敏感。然而，它的可行性定量分析的蛋白质能量尚未得到评估。在这里，我们评估A（230）作为结构探针，以确定蛋白质的热力学稳定性和去折叠动力学。利用大肠杆菌麦芽糖结合蛋白（MBP）和大肠杆菌E.大肠杆菌核糖核酸酶H（RNase H）作为我们的模型蛋白，我们用A（230）监测了它们在尿素和氯化胍中的解折叠。两种蛋白质在化学变性剂中展开时，观察到A（230）的显著变化。通过测量在不同浓度的变性剂中A（230）的变化，成功地确定了全局稳定性。此外，通过监测变性条件下A（230）的变化来研究解折叠动力学。测定结果与圆二色谱法测定结果一致。与CD不同，A（230）允许我们用UV酶标仪在96孔微量滴定板中监测蛋白质解折叠。我们的研究结果表明，A（230）是一个有效的和方便的结构探针，以确定蛋白质的热力学稳定性和去折叠动力学具有许多潜在的应用。(C)2009 Elsevier Inc. All rights reserved.
Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A(230)) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A(230) as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A(230). Significant changes in A(230) were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A(230) in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A(230) under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A(230) allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A(230) is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications. (C) 2009 Elsevier Inc. All rights reserved.