Jazayeri Fatemeh S, Amininasab Mehriar, Hosseinkhani Saman
Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, IRAN.
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IRAN.
PLoS One. 2017 Jul 3;12(7):e0180667. doi: 10.1371/journal.pone.0180667. eCollection 2017.
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.
荧光素酶是生物发光过程中发光的关键成分。由于其热稳定性较低，这种酶在生物技术中的广泛且有益的应用受到了限制。在此我们报告了与298 K温度相比，加热至高于熔点（Tm）对荧光素酶的结构和动力学性质的影响。通过这种方式，我们证明了在325 K时，游离酶的N结构域和C结构域之间的氢键数量增加。在325 K时结构域间氢键增加了三个，这表明结构域间的接触得到了加强。K529和D422之间同时出现强盐桥和氢键以及R533和E389之间存在概率增加，从机制上解释了N结构域和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。此外，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的溶剂可及表面积（SASA）显著降低。我们研究中的主成分分析（PCA）表明，酶在加热时动力学行为发生了变化，这主要源于运动模式的改变以及这些运动相对于298 K的相关程度。这些发现可以解释为什么随着时间的推移，酶的加热或蛋白质构象的热波动会降低荧光素酶的活性，这是热功能失活的一种可能机制。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。
