Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase.
Structural and dynamical insight into thermally induced functional inactivation of firefly luciferase.
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DOI：
10.1371/journal.pone.0180667
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Hosseinkhani S
中科院分区：
文献类型：
--
作者：
Jazayeri FS;Amininasab M;Hosseinkhani S
荧光素酶是生物发光过程中产生光的关键成分。该酶热稳定性差，限制了其在生物技术中的广泛应用。在这里，我们报告的影响，加热到Tm以上的荧光素酶的结构和动力学性质相比，在298 K的温度。通过这种方式，我们证明了在325 K下，游离酶的N-和C-结构域之间的氢键数量增加。在325 K时增加了三个畴间氢键，表明畴间接触得到加强。K529和D422之间同时出现强盐桥和氢键以及R533和E389之间存在概率的增加可以从机理上解释N-和C-结构域之间更强的接触。诱变研究通过实验证明了K529和D422的重要性。还观察到参与活性位点区域的实验上重要的残基K529、D422和T343的SASA的显著降低。主成分分析（PCA）结果表明，酶的动力学行为在升温过程中发生了变化，这主要是由于相对于298 K，酶的运动模式和运动幅度发生了变化。这些发现可以解释为什么酶的加热或蛋白质构象的热波动会随着时间的推移降低荧光素酶活性，这可能是热功能失活的一种机制。根据这些结果，我们提出了两种提高功能性荧光素酶热稳定性的策略。
Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase.