Contour length and refolding rate of a small protein controlled by engineered disulfide bonds
Contour length and refolding rate of a small protein controlled by engineered disulfide bonds
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DOI：
10.1529/biophysj.106.091561
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发表时间：
2007-01-01
影响因子：
3.4
通讯作者：
Fernandez, Julio M.
中科院分区：
文献类型：
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作者：
Ainavarapu, Rama Koti;Brujic, Jasna;Fernandez, Julio M.
蛋白质中二硫键的引入产生了额外的机械障碍，限制了单分子力谱测量的未折叠轮廓长度(即最大伸展长度)。在这里，我们在人类心脏肌动蛋白模块(I27)的四个不同位置设计了单一的二硫键，以控制轮廓长度，同时保持到过渡状态的距离不变。这使得研究几个生物学上重要的参数成为可能。首先，我们能够精确地确定展开前过渡态的端到端长度(53埃)，这比核磁共振光谱获得的蛋白质的端到端长度(43埃)要长。其次，从五种不同的方法测得的每个氨基酸的轮廓长度(4.0+/-0.2埃)比从晶体结构得到的端到端的长度(3.6埃)要长。我们对轮廓长度的测量考虑了多肽链的所有内部自由度，而结晶学测量的是“冻结的”蛋白质结构中的端到端长度。此外，轮廓长度的控制以及因此在到达二硫键(N)之前分解的氨基酸数量的控制有助于测试链长与折叠时间(tau(F))的关系。我们发现，当lambda=4.4时，与n(Lambda)成正比的幂定律标度tau(F)和与n(0.6)的指数标度都符合数据范围，以支持不同的蛋白质折叠场景。
The introduction of disulfide bonds into proteins creates additional mechanical barriers and limits the unfolded contour length (i.e., the maximal extension) measured by single-molecule force spectroscopy. Here, we engineer single disulfide bonds into four different locations of the human cardiac titin module (I27) to control the contour length while keeping the distance to the transition state unchanged. This enables the study of several biologically important parameters. First, we are able to precisely determine the end-to-end length of the transition state before unfolding (53 angstrom), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 angstrom). Second, the measured contour length per amino acid from five different methods (4.0 +/- 0.2 angstrom) is longer than the end-to-end length obtained from the crystal structure (3.6 angstrom). Our measurement of the contour length takes into account all the internal degrees of freedom of the polypeptide chain, whereas crystallography measures the end-to-end length within the "frozen'' protein structure. Furthermore, the control of contour length and therefore the number of amino acids unraveled before reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (tau(F)). We find that both a power law scaling tau(F) proportional to n(lambda) with lambda = 4.4, and an exponential scaling with n(0.6) fit the data range, in support of different protein-folding scenarios.