Iron-sulfur cluster biosynthesis -: Biochemical characterization of the conformational dynamics of Thermotoga maritima IscU and the relevance for cellular cluster assembly
Iron-sulfur cluster biosynthesis -: Biochemical characterization of the conformational dynamics of Thermotoga maritima IscU and the relevance for cellular cluster assembly
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DOI：
10.1074/jbc.m312051200
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发表时间：
2004-03-12
影响因子：
4.8
通讯作者：
Cowan, JA
中科院分区：
文献类型：
--
作者：
Mansy, SS;Wu, SP;Cowan, JA
重要的是了解铁硫支架IscU的功能特性是这类蛋白质的结构和动力学知识。（2002）海洋热生菌（Thermotoga martima IscU）的结构表征（Mansy, s.s S, Wu， G.）化学，277,21397-21404)和高分辨率核磁共振（Bertini， I., Cowan， J. A., Del Bianco， C., Luchinat， C., and Mansy， S. S. (2003) J. Mol. Biol, 331, 907-924）得出的数据表明了高度的二级结构。然而，后者也揭示了IscU存在于两个或多个不同构象之间的动态平衡中，可能以熔融球态存在。在此，我们通过近紫外圆二色性、1-苯胺-8-萘磺酸结合、展开自由能、流体动力半径测量和有限的胰蛋白酶消化进一步表征了T. maritima IscU的熔融球特性。数据显示不寻常的动态行为不完全符合典型的蛋白质状态，如完全折叠，完全展开，或熔融球。例如，近紫外CD光谱和流体动力学半径测量支持稳定第三纪褶皱的存在，而其他数据则不太清楚地解释，可能被视为与熔融球或完全折叠状态相一致。然而，所有的数据都与我们之前的假设一致，即蛋白质采样多个离散的三级构象，其中这些结构转变发生在“缓慢”的时间尺度上。为了描述这样的蛋白质，我们引入了术语多重离散构象。
Important for the understanding of the functional properties of the iron-sulfur scaffold IscU is knowledge of the structure and dynamics of this protein class. Structural characterization of Thermotoga maritima IscU by CD (Mansy, S. S., Wu, G., Surerus, K. K., and Cowan, J. A. (2002) J. Biol. Chem. 277, 21397-21404) and high resolution NMR (Bertini, I., Cowan, J. A., Del Bianco, C., Luchinat, C., and Mansy, S. S. (2003) J. Mol. Biol. 331, 907-924) yielded data indicating a high degree of secondary structure. However, the latter also revealed IscU to exist in a dynamic equilibrium between two or more distinct conformations, possibly existing in a molten globule state. Herein, we further characterize the molten globule characteristics of T. maritima IscU by near-ultraviolet circular dichroism, 1-anilino-8-naphthalenesulfonic acid binding, free energy of unfolding, hydrodynamic radius measurements, and limited tryptic digestion. The data suggest unusual dynamic behavior that is not fully consistent with typical protein states such as fully folded, fully unfolded, or molten globule. For instance, the existence of a stable tertiary fold is supported by near-UV CD spectra and hydrodynamic radius measurements, whereas other data are less clearly interpretable and may be viewed as consistent with either a molten globule or fully folded state. However, all of the data are consistent with our previous hypothesis of a protein sampling multiple discrete tertiary conformations in which these structural transitions occur on a "slow" time scale. To describe such proteins, we introduce the term multiple discrete conformers.