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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Biophysical characterization of higher plant Rubisco activase
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Biophysical characterization of higher plant Rubisco activase

机译：高等植物Rubisco活化酶的生物物理特性

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摘要

Rubisco activase (Rca) is a chaperone-like protein of the AAA + family, which uses mechano-chemical energy derived from ATP hydrolysis to release tightly bound inhibitors from the active site of the primary carbon fixing enzyme ribulose 1,5-bisphosphate oxygenase/carboxylase (Rubisco). Mechanistic and structural investigations of Rca have been hampered by its exceptional thermolability, high degree of size polydispersity and propensity towards subunit aggregation. In this work, we have characterized the thermal stability and self-association behavior of recombinant Rca preparations, and have developed ligand screening methods. Thermal denaturation profiles generated by circular dichroism indicate that creosote and tobacco short-form Rcas are the most stable proteins examined, with an estimated mid-point temperature of 45-47 C for protein denaturation. We demonstrate that ADP provides a higher degree of stabilization than ATP, that magnesium ions have a small stabilizing effect on ATP-bound, but a significant destabilizing effect on ADP-bound Rca, and that phosphate provides weak stabilization of the ADP-bound form of the protein. A dimeric species was identified by size-exclusion chromatography, suggesting that the two-subunit module may comprise the basic building block for larger assemblies. Evidence is provided that chromatographic procedures reflect non-equilibrium multimeric states. Dynamic light scattering experiments performed on nucleotide-bearing Rca support the notion that several larger, highly polydisperse assembly states coexist over a broad concentration range. No significant changes in aggregation are observed upon replacement of ADP with ATP. However, in the absence of nucleotides, the major protein population appears to consist of a monodisperse oligomer smaller than a hexamer. ? 2012 Elsevier B.V. All rights reserved.
机译：Rubisco活化酶（Rca）是AAA +家族的伴侣蛋白，它利用ATP水解产生的机械化学能从碳固定酶核糖1,5-双磷酸加氧酶/羧化酶（Rubisco）。 Rca出色的可热性，高度的粒度多分散性和对亚基聚集的倾向阻碍了Rca的机理和结构研究。在这项工作中，我们表征了重组Rca制剂的热稳定性和自缔合行为，并开发了配体筛选方法。圆二色性产生的热变性图表明，杂酚油和烟草短型Rcas是最稳定的蛋白质，估计蛋白质变性的中点温度为45-47C。我们证明，ADP比ATP提供更高的稳定性，镁离子对ATP结合具有较小的稳定作用，但对与ADP结合的Rca具有显着的去稳定作用，并且磷酸盐提供的ADP结合形式的磷酸化作用较弱。蛋白质。通过尺寸排阻色谱法鉴定了二聚体物种，表明该二亚基模块可以构成用于较大装配体的基本构件。提供了色谱程序反映非平衡多聚体状态的证据。在带有核苷酸的Rca上进行的动态光散射实验支持以下观点：在较大的浓度范围内，多个较大的高度多分散的组装状态共存。用ATP代替ADP后，未观察到聚集的显着变化。但是，在没有核苷酸的情况下，主要的蛋白质种群似乎由比六聚体小的单分散低聚物组成。 ？ 2012 Elsevier B.V.保留所有权利。

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