Probing the Internal pH and Permeability of a Carboxysome Shell.
Probing the Internal pH and Permeability of a Carboxysome Shell.
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DOI：
10.1021/acs.biomac.2c00781
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发表时间：
2022-10-10
影响因子：
6.2
通讯作者：
Liu, Lu-Ning
中科院分区：
文献类型：
--
作者：
Huang, Jiafeng;Jiang, Qiuyao;Yang, Mengru;Dykes, Gregory F.;Weetman, Samantha L.;Xin, Wei;He, Hai-Lun;Liu, Lu-Ning
碳氧体是蓝细菌和许多变形菌中基于蛋白质的纳米级细胞器，其将关键的CO2固定酶核酮糖-1，5-二磷酸羧化酶/加氧酶（Rubisco）和碳酸酐酶（CA）封装在多面体蛋白质壳内。羧基体的内在自组装和结构特征以及蛋白质壳的半渗透性为羧基体内CO2的积累和增强羧基化提供了基础。在这里，我们开发了一种方法来确定内部的pH值条件和无机碳积累的α-羧基壳来自化能自养蛋白杆菌那不勒斯盐硫杆菌和评估壳的渗透性。通过在大肠杆菌中产生的空α-羧基体壳内掺入pH报告基因pHluorin 2，我们探测了有和没有CA的蛋白质壳的内部pH。我们的体内和体外结果表明，α-羧基体壳的内部pH值低于细胞质pH值和缓冲液pH值，以及响应外部环境变化的内部pH值的调节，表明壳对碳酸氢根离子和质子的渗透性。我们进一步确定了α-羧基体壳内15 mM的饱和HCO 3-浓度，并显示了CA介导的内部CO2水平的增加。揭示carboxysomes的内部理化微环境是至关重要的理解carboxysomal壳的渗透性和增强的Rubisco羧化carboxysomes内的机制。这些基础知识可以告知重新编程羧基体以改善代谢并招募外源酶以增强催化性能。
The carboxysome is a protein-based nanoscale organelle in cyanobacteria and many proteobacteria, which encapsulates the key CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a polyhedral protein shell. The intrinsic self-assembly and architectural features of carboxysomes and the semipermeability of the protein shell provide the foundation for the accumulation of CO2 within carboxysomes and enhanced carboxylation. Here, we develop an approach to determine the interior pH conditions and inorganic carbon accumulation within an α-carboxysome shell derived from a chemoautotrophic proteobacterium Halothiobacillus neapolitanus and evaluate the shell permeability. By incorporating a pH reporter, pHluorin2, within empty α-carboxysome shells produced in Escherichia coli, we probe the interior pH of the protein shells with and without CA. Our in vivo and in vitro results demonstrate a lower interior pH of α-carboxysome shells than the cytoplasmic pH and buffer pH, as well as the modulation of the interior pH in response to changes in external environments, indicating the shell permeability to bicarbonate ions and protons. We further determine the saturated HCO3– concentration of 15 mM within α-carboxysome shells and show the CA-mediated increase in the interior CO2 level. Uncovering the interior physiochemical microenvironment of carboxysomes is crucial for understanding the mechanisms underlying carboxysomal shell permeability and enhancement of Rubisco carboxylation within carboxysomes. Such fundamental knowledge may inform reprogramming carboxysomes to improve metabolism and recruit foreign enzymes for enhanced catalytical performance.
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