Ligand binding modulates the mechanical stability of dihydrofolate reductase
Ligand binding modulates the mechanical stability of dihydrofolate reductase
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DOI：
10.1529/biophysj.105.062034
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发表时间：
2005-11-01
影响因子：
3.4
通讯作者：
Fernandez, JM
中科院分区：
文献类型：
--
作者：
Ainavarapu, RK;Li, LY;Fernandez, JM
我们使用单分子力光谱证明，二氢叶酸还原酶（DHFR）的机械稳定性是由配体结合调制。在没有结合配体的情况下，DHFR以非常低的力延伸，平均27 pN，没有任何特征性的机械指纹。相比之下，在存在微摩尔浓度的配体甲氨蝶呤、烟酰胺腺嘌呤磷酸二氢盐或二氢叶酸盐的情况下，需要高得多的力（分别为82 +/- 18 pN、98 +/- 15 pN和83 +/- 16 pN），并且在力-延伸曲线中观察到特征指纹。由这些配体引发的增加的机械稳定性不是加和的。我们的研究结果解释了DHFR的降解速率大幅降低，在其配体的存在下。我们的观察结果支持这样的观点，即三磷酸腺苷依赖性蛋白酶降解蛋白质的限速步骤是靶蛋白的机械展开。
We use single-molecule force spectroscopy to demonstrate that the mechanical stability of the enzyme dihydrofolate reductase ( DHFR) is modulated by ligand binding. In the absence of bound ligands, DHFR extends at very low forces, averaging 27 pN, without any characteristic mechanical fingerprint. By contrast, in the presence of micromolar concentrations of the ligands methotrexate, nicotinamide adenine dihydrogen phosphate, or dihydrofolate, much higher forces are required (82 +/- 18 pN, 98 +/- 15 pN, and 83 +/- 16 pN, respectively) and a characteristic fingerprint is observed in the force-extension curves. The increased mechanical stability triggered by these ligands is not additive. Our results explain the large reduction in the degradation rate of DHFR, in the presence of its ligands. Our observations support the view that the rate-limiting step in protein degradation by adenosine triphosphate-dependent proteases is the mechanical unfolding of the target protein.