Mechanical and chemical unfolding of a single protein: A comparison
Mechanical and chemical unfolding of a single protein: A comparison
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DOI：
10.1073/pnas.96.7.3694
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发表时间：
1999-03-30
影响因子：
11.1
通讯作者：
Fernandez, JM
中科院分区：
文献类型：
--
作者：
Carrion-Vazquez, M;Oberhauser, AF;Fernandez, JM
原子力显微镜(AFM)对蛋白质结构域的机械分解仅仅是一项技术壮举，还是对其展开的真实测量？通过设计一个由相同Ig模块的串联重复序列组成的蛋白质，我们能够获得关于单个蛋白质结构域的显式AFM数据，该数据可以准确地外推到零力。我们将其与外推至0M变性剂的未系链模块的化学展开速率进行比较。这两种方法得到的展开速率是相同的。此外，无论采用哪种方法，蛋白质的去折叠过渡态都出现在折叠路径上的同一位置，这些结果表明，AFM对单个蛋白质的机械去折叠确实反映了传统去折叠实验中观察到的相同事件，这为AFM在单分子水平上测量折叠反应开辟了道路。单分子AFM记录还有一个额外的优势，那就是它们定义了反应坐标，并揭示了在没有化学变性剂的情况下无法观察到的罕见的展开事件。
Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method, These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments, The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants.