Nonequilibrium single molecule protein folding in a coaxial mixer
Nonequilibrium single molecule protein folding in a coaxial mixer
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DOI：
10.1529/biophysj.107.127431
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发表时间：
2008-07-01
影响因子：
3.4
通讯作者：
Weiss, Shimon
中科院分区：
文献类型：
--
作者：
Hamadani, Kambiz M.;Weiss, Shimon
我们已经开发出一种连续流混合装置，适用于监测生物构象反应，在单分子水平的响应时间类似于10毫秒下的单分子流动条件。它的同轴几何形状允许样品流体的三维流体动力学聚焦到衍射限制的尺寸，其中扩散混合是快速和有效的。基于毛细管的设计能够在实验室内快速构建混合器，而不需要昂贵的基于光刻的微加工设施。使用颗粒状二氧化硅颗粒在线过滤样品流体几乎消除了堵塞，并将每个设备的使用寿命延长至数月。在这篇文章中，要确定的距离到时间的传递函数和仪器的响应函数的设备，我们使用荧光互相关光谱测速和有限元流体动力学模拟表征其流体流动和混合特性。然后，我们将混合器应用于胰凝乳蛋白酶抑制剂蛋白2的单分子FRET蛋白质折叠研究。通过瞬时填充未折叠状态的糜蛋白酶抑制剂蛋白2（CI2）在非平衡体外重折叠条件下，我们在空间和时间上解决变性剂依赖的非特异性崩溃的未折叠状态的障碍限制折叠过渡CI2。我们的研究结果是一致的，与以前的CI2混合的结果，发现证据的异构展开状态组成的顺式和反式脯氨酸构象。
We have developed a continuous-flow mixing device suitable for monitoring bioconformational reactions at the single-molecule level with a response time of similar to 10 ms under single-molecule flow conditions. Its coaxial geometry allows three-dimensional hydrodynamic focusing of sample fluids to diffraction-limited dimensions where diffusional mixing is rapid and efficient. The capillary-based design enables rapid in-lab construction of mixers without the need for expensive lithography-based microfabrication facilities. In-line filtering of sample fluids using granulated silica particles virtually eliminates clogging and extends the lifetime of each device to many months. In this article, to determine both the distance-to-time transfer function and the instrument response function of the device we characterize its fluid flow and mixing properties using both fluorescence cross-correlation spectroscopy velocimetry and finite element fluid dynamics simulations. We then apply the mixer to single molecule FRET protein folding studies of Chymotrypsin Inhibitor protein 2. By transiently populating the unfolded state of Chymotrypsin Inhibitor Protein 2 ( CI2) under nonequilibrium in vitro refolding conditions, we spatially and temporally resolve the denaturant-dependent nonspecific collapse of the unfolded state from the barrier-limited folding transition of CI2. Our results are consistent with previous CI2 mixing results that found evidence for a heterogeneous unfolded state consisting of cis- and trans-proline conformers.