Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein
Linked folding and anion binding of the Bacillus subtilis ribonuclease P protein
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DOI：
10.1021/bi002078y
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发表时间：
2001-03-06
期刊：
影响因子：
2.9
通讯作者：
Oas, TG
中科院分区：
文献类型：
--
作者：
Henkels, CH;Kurz, JC;Oas, TG
核糖核酸酶P(RNaseP)是负责前体tRNA转录本5‘-成熟的内切核酸酶。在细菌中，RNaseP由一个催化的RNA亚基和一个相关的蛋白质亚基组成，它增强了全酶的底物专一性。我们已经开始了对枯草芽孢杆菌核糖核酸酶P(P蛋白)蛋白亚基生物物理性质的研究，目的是为了了解RNase P全酶组装的热力学。根据圆二色谱和核磁共振研究，P蛋白主要在中性pH的10 mM碳酸氢钠中展开，因此具有典型的“内在非结构”蛋白质的几个特征。此外，当加入各种小分子阴离子时，P蛋白折叠成其天然的α/β结构。阴离子诱导的折叠最好地归因于这些阴离子与蛋白质的折叠状态的结合，并提出了一个模型来描述观察到的紧密耦合的折叠和结合现象。当渗透分子三甲胺N-氧化物(TMAO)加入时，P蛋白也经历了协同折叠转变。用二态折叠模型描述TMAO滴定数据，测得P蛋白37℃时的K折叠平衡常数为0.0071+/-0.0005。因此，在负离子诱导的P蛋白折叠中观察到的折叠和结合平衡可以解偶联来确定阴离子配体的内在结合亲和力(K-a‘s)。还提出了渗透分子诱导的P蛋白折叠构象和配体诱导的折叠构象在结构上相似的证据。
Ribonuclease P (RNase P) is the endoribonuclease responsible for the 5'-maturation of precursor tRNA transcripts. In bacteria, RNase P is composed of a catalytic RNA subunit and an associated protein subunit that enhances the substrate specificity of the holoenzyme. We have initiated a study of the biophysical properties of the protein subunit from Bacillus subtilis RNase P (P protein) toward the goal of understanding the thermodynamics of RNase P holoenzyme assembly. The P protein is predominantly unfolded in 10 mM sodium cacodylate at neutral pH based on circular dichroism and NMR studies and therefore has several characteristics typical of "intrinsically unstructured" proteins. Furthermore, the P protein folds to its native alpha/beta structure upon addition of various small molecule anions. Anion-induced folding is best attributed to the binding of these anions to the folded state of the protein, and a model is presented which describes the observed tightly coupled folding and binding phenomena. The P protein also undergoes a cooperative folding transition upon addition of the osmolyte trimethylamine N-oxide (TMAO). The equilibrium constant of folding (K-fold) at 37 degreesC for the P protein was determined to be 0.0071 +/- 0.0005 using a two-state folding model to describe the TMAO titration data. Thus, the folding and binding equilibria observed in the anion-induced folding of the P protein can be uncoupled to determine the intrinsic binding affinities (K-a's) of the anionic ligands. Evidence that the osmolyte-induced and the ligand-induced folded conformations of the P protein are structurally similar is also presented.