The Intrinsically Disordered RNR Inhibitor Sml1 Is a Dynamic Dimer
The Intrinsically Disordered RNR Inhibitor Sml1 Is a Dynamic Dimer
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DOI：
10.1021/bi801040b
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发表时间：
2008-12-16
期刊：
影响因子：
2.9
通讯作者：
Kragelund, Birthe B.
中科院分区：
文献类型：
--
作者：
Danielsson, Jens;Liljedahl, Leena;Kragelund, Birthe B.
Sml1 是酿酒酵母中的一种小核糖核苷酸还原酶 (RNR) 调节蛋白，可结合并抑制 RNR 激活。 N-15 标记的 Sml1（104 个残基）以及截短变体（残基 50-104）的 NMR 研究可以表征其分子特性。 Sml1 属于本质上无序的蛋白质类别，具有高度动态性和很少的稳定结构。早期关于 Sml1 二聚体结构的建议得到了证实，并且根据平移扩散 NMR 测量，可以确定 4°C 下的二聚解离常数为 0.1 mM。 Sml1 单体形式的流体动力学半径被确定为 23.4 埃，对应于球状蛋白和卷曲蛋白之间的蛋白大小。二聚体的形成导致流体动力学半径为 34.4 埃。观察到的化学位移显示与之前的研究一致，两个片段具有瞬时螺旋结构，残基4-20和60-86，并且松弛研究清楚地表明这些片段中的运动受限。连接到 C14 的自旋标记显示与残基 60-70 和 85-95 的长程相互作用，表明 N 端结构域折叠到 C 端结构域上。重要的是，蛋白酶降解研究与质谱分析相结合表明，N 端结构域在 C 端区域之前被降解，因此可以作为功能上重要的 C 端区域免受蛋白水解的保护。脏的形成与结构的显着诱导无关，但发现可以提供进一步的保护以防止蛋白水解。我们认为，这种分子屏蔽和保护重要功能结构免遭功能不重要位点降解可能是其他天然无序蛋白质的一般属性。
Sml1 is a small ribonucleotide reductase (RNR) regulatory protein in Saccharomyces cerevisiae that binds to and inhibits RNR activation. NMR studies of N-15-labeled Sml1 (104 residues), as well as of a truncated variant (residues 50-104), have allowed characterization of their molecular properties. Sml1 belongs to the class of intrinsically disordered proteins with a high degree of dynamics and very little stable structure. Earlier suggestions for a dimeric structure of Sml1 were confirmed, and from translation diffusion NMR measurements, a dimerization dissociation constant of 0.1 mM at 4 degrees C could be determined. The hydrodynamic radius for the monomeric form of Sml1 was determined to be 23.4 angstrom, corresponding to a protein size between those of a globular protein and a coil. Formation of a dimer results in a hydrodynamic radius of 34.4 angstrom. The observed chemical shifts showed in agreement with previous studies two segments with transient helical structure, residues 4-20 and 60-86, and relaxation studies clearly showed restricted motion in these segments. A spin-label attached to C14 showed long-range interactions with residues 60-70 and 85-95, suggesting that the N-terminal domain folds onto the C-terminal domain. Importantly, protease degradation studies combined with mass spectrometry indicated that the N-terminal domain is degraded before the C-terminal region and thus may serve as a protection against proteolysis of the functionally important C-terminal region. Dirtier formation was not associated with significant induction of structure but was found to provide further protection against proteolysis. We propose that this molecular shielding and protection of vital functional structures from degradation by functionally unimportant sites may be a general attribute of other natively disordered proteins.