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首页> 外文期刊>Biochemistry >Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29
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Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29

机译：结合折叠在古细菌RNase P蛋白RPP21和RPP29相互作用中的热力学。

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We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg~(2+)-dependent cleavage of the 5' leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5?RPP30 and RPP21-RPP29. Nuclear magnetic resonance studies of the Pyrococcus f uriosus RPP21 and RPP29 proteins in their free and complexed states provided evidence of significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, and pH values, in buffers with varying ionization potentials, and with a foldingdeficient RPP21 point mutant. These experiments revealed a negative heat capacity change (ΔC_p), nearly twice that predicted from surface accessibility calculations, a strong salt dependence for the interaction, and proton release at neutral pH, but a small net contribution from these to the excess ΔC_p. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess ΔC_p. These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies.
机译：我们已使用等温滴定热法（ITC）来识别和描述古细菌核糖核酸酶P（RNase P）的两个蛋白质亚基之间相互作用的结合偶联平衡。在生活的所有三个领域中，RNase P是一种核糖核蛋白复合物，主要负责在tRNA成熟过程中催化前体tRNA的5'前导序列的Mg〜（2+）依赖性切割。在古细菌中，已显示出RNase P由一种催化性RNA和多达五种蛋白质组成，其中四种在不存在RNA的情况下结合为两种功能性异源二聚体，即POP5？RPP30和RPP21-RPP29。游离火球菌RPP21和RPP29蛋白处于游离状态和复杂状态的核磁共振研究提供了结合后显着蛋白质折叠的证据。 ITC实验是在温度，离子强度和pH值的范围内，具有不同电离电势和折叠缺陷的RPP21点突变体的缓冲液中进行的。这些实验揭示了负热容变化（ΔC_p），几乎是表面可及性计算所预测的两倍，相互作用的强烈盐依赖性以及在中性pH下质子释放，但是这些对过量ΔC_p的净贡献很小。我们基于结构和光谱数据考虑了蛋白质折叠和界面水分子埋藏的潜在贡献。我们得出的结论是，结合偶联的蛋白质折叠可能是过量ΔC_p的重要部分。这些发现为偶联平衡提供了新颖的结构和热力学见解，从而使大分子组装具有特异性。

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