Background Mitochondria are unique organelles that are found in most eukaryotic cells. The main role of the mitochondria is to produce ATP. The nuclear genome encoded proteins Cbs1 and Cbs2 are located at the mitochondrial inner membrane and are reported to be essential for the translation of mitochondrial cytochrome b mRNA. Genetic studies show that Cbs2 protein recognizes the 5′ untranslated leader sequence of mitochondrial cytochrome b mRNA. However, due to a lack of biochemical and structural information, this biological process remains unclear. To investigate the structural characteristics of how Saccharomyces cerevisiae (S. cerevisiae) Cbs2 tethers cytochrome b mRNA to the mitochondrial inner membrane, a preliminary X-ray crystallographic study was carried out and is reported here. Methods The target gene from S. cerevisiae was amplified by polymerase chain reaction. The PCR fragment was digested by the NdeI and XhoI restriction endonucleases and then inserted into expression vector p28. After sequencing, the plasmid was transformed into Escherichia coli C43 competent cells. The selenomethionine derivative Cbs2 protein was overexpressed using M9 medium based on a methionine-biosynthesis inhibition method. The protein was first purified to Ni2+-nitrilotriacetate affinity chromatography and then further purified by Ion exchange chromatography and Gel-filtration chromatography. The purified Se-Cbs2 protein was concentrated to 10 mg/mL. The crystallization trials were performed using the sitting-drop vapor diffusion method at 16 °C. The complete diffraction data was processed and scaled with the HKL2000 package and programs in the CCP4 package, respectively. Results Cbs2 from S. cerevisiae was cloned, prokaryotic expressed and purified. The analysis of the size exclusion chromatography showed that the Cbs2 protein peaked at a molecular weight of approximately 90 KDa. The crystal belonged to the space group C2, with unit-cell parameters of a = 255.11, b = 58.10, c = 76.37, and β = 95.35°. X-ray diffraction data was collected at a resolution of 2.7 Å. The Matthews coefficient and the solvent content were estimated to be 3.22 Å 3 Da-1 and 61.82%, respectively. Conclusions In the present study Cbs2 from S. cerevisiae was cloned, expressed, purified, and crystallized for structural studies. The molecular weight determination results indicated that the biological assembly of Cbs2 may be a dimer.The preliminary X-ray crystallographic studies indicated the presence of two Cbs2 molecules in the asymmetric unit. This study will provide an experimental basis for exploring how Cbs2 protein mediates cytochrome b synthesis.

中文翻译：

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酿酒酵母线粒体膜相关蛋白 Cbs2 的表达、纯化、结晶和初步 X 射线晶体学研究

背景 线粒体是在大多数真核细胞中发现的独特细胞器。线粒体的主要作用是产生ATP。核基因组编码的蛋白质 Cbs1 和 Cbs2 位于线粒体内膜，据报道对线粒体细胞色素 b mRNA 的翻译至关重要。遗传学研究表明，Cbs2 蛋白识别线粒体细胞色素 b mRNA 的 5' 非翻译前导序列。然而，由于缺乏生化和结构信息，这一生物学过程仍不清楚。为了研究酿酒酵母 (S. cerevisiae) Cbs2 如何将细胞色素 b mRNA 束缚到线粒体内膜的结构特征，进行了初步的 X 射线晶体学研究，并在此报道。方法 目的基因来自 S. 通过聚合酶链反应扩增酿酒酵母。PCR 片段被 NdeI 和 XhoI 限制性内切酶消化，然后插入表达载体 p28。测序后，将质粒转化到大肠杆菌C43感受态细胞中。基于蛋氨酸生物合成抑制方法，使用M9培养基过表达硒代蛋氨酸衍生物Cbs2蛋白。蛋白质首先通过Ni2+-次氮基三乙酸亲和层析纯化，然后通过离子交换层析和凝胶过滤层析进一步纯化。将纯化的 Se-Cbs2 蛋白浓缩至 10 mg/mL。结晶试验在 16 °C 下使用坐滴蒸汽扩散法进行。完整的衍射数据分别使用 HKL2000 包和 CCP4 包中的程序进行处理和缩放。结果克隆、原核表达和纯化酿酒酵母Cbs2。尺寸排阻色谱分析表明，Cbs2 蛋白在分子量约为 90 KDa 处达到峰值。该晶体属于空间群C2，晶胞参数a = 255.11，b = 58.10，c = 76.37，β = 95.35°。以 2.7 Å 的分辨率收集 X 射线衍射数据。Matthews 系数和溶剂含量估计分别为 3.22 Å 3 Da-1 和 61.82%。结论 在本研究中，来自酿酒酵母的 Cbs2 被克隆、表达、纯化和结晶用于结构研究。分子量测定结果表明Cbs2的生物组装可能是二聚体。初步的 X 射线晶体学研究表明在不对称单元中存在两个 Cbs2 分子。本研究将为探索Cbs2蛋白如何介导细胞色素b合成提供实验基础。