Resolving dynamics and function of transient states in single enzyme molecules
Resolving dynamics and function of transient states in single enzyme molecules
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DOI：
10.1038/s41467-020-14886-w
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发表时间：
2020-03-06
影响因子：
16.6
通讯作者：
Seidel, Claus A. M.
中科院分区：
文献类型：
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作者：
Sanabria, Hugo;Rodnin, Dmitro;Seidel, Claus A. M.
我们使用一个混合荧光光谱工具包来监测T4溶菌酶（T4 L）的行动，通过解开的构象状态的动力学和动态相互作用。特别是，通过结合单分子和合奏多参数荧光检测，EPR光谱，诱变，FRET定位和筛选，以及其他生物化学和生物物理工具，我们表征了三个短暂的构象状态的ns-ms时间尺度。使用33 FRET衍生的距离集，筛选可用的T4 L结构，揭示T4 L在溶液中主要采用已知的开放和关闭状态在4 μ s的交换。一个新发现的次要状态，未公开的，目前，超过500个晶体结构的T4 L和采样在230 μ s，可能积极参与催化的产物释放步骤。所提出的荧光光谱工具包可能会加速动态结构生物学的发展，通过识别瞬时构象状态，是非常丰富的生物学和关键的酶促反应。
We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 mu s. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 mu s, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.