MOLECULAR ARCHITECTURE OF UQH2: CYT C2 OXIDOREDUCTASE

UQH2：Cyt c2的功能和分子结构 球状乳杆菌氧化还原酶的研究采用组合方法。 利用分子遗传学、生物化学和生物物理学。我们正在修改 编码该酶的基因，并分析功能和 蛋白质中特定变化的结构后果。通过使用 新的蛋白质结构预测算法，以及来自 在导致抑制剂耐药性的病变的残留水平上进行映射， 我们提出了一个8-跨膜的Felix模型，并定位了 两个对苯二酚反应催化中心。我们已经开始修改残留物 在可能在催化中起重要作用的假定位置。我们的目标是 通过详细的生物物理绘制特定残留物的贡献图 通过定点突变或随机突变产生的突变的研究，以及 利用这些信息来分析 网站。我们正在使用类似的方法来覆盖结构，组装， 和酶的拓扑结构。细胞色素b的拓扑学研究 使用PhoA融合技术的亚基支持我们的8膜 Helic模型，并利用球形红曲霉进行了防治试验 反应中心L-亚尼特证明PHOA融合的有效性 技术 我们已经完成了编码FBC操纵子的DNA测序 三种多肽(含亚基的双细胞色素b、细胞色素c1和2Fe.2S) 并推导出氨基酸序列。我们已经开发出一种预备性的 允许大规模提纯一种活性较低的酶的协议， 已经完成了初步的生化和生物物理特征- 埃里克化。亚基的初步N-末端测序 确认了来自DNA测序的数据，并显示了一些帖子 转换处理。通过在适当的位置引入卡那霉素盒 在fbcf基因中，我们已经制备了一株缺乏功能性fbc的菌株。 不能光合作用生长的操纵子(pho-)。 与FBC操纵子的互补导致修复 Pho+表型。我们已经制造了一些定点突变体，并且已经 开始了Cinetic和Cinetic的详细生物物理表征 热力学参数。我们还选择了具有抗性的抑制剂 突变株，并对其进行了初步表征 耐药菌株。 我们已经建立了一个计算机辅助荧光视频的装置 帮助筛选和初步鉴定突变体的成像技术 菌株。开发的软件允许我们选择具有特定基因的菌株 使用图像的虚假颜色编码的表型属性，以及测量 单个菌落的诱导动力学。

The function and molecular architecture of the UQH2:cyt c2 oxidoreductase of R. spheroides are being studied by a combined approach using molecular genetics, biochemistry and biophysics. We are modifying the genes coding for the enzyme, and assaying the functional and structural consequences of specific changes in the proteins. By using new protein structural prediction algorithms, and information from the mapping at the residue level of lesions leading to inhibitor resistance, we have proposed an 8-transmembrane felix model, and have located the two quinone reactive catalytic sites. We have started to modify residues at the putative sites which might be important in catalysis. Our goal is to map the contribution of specific residues by detailed biophysical studies of mutants generated by site-specific or random mutagenesis, and to use this information to analyse mechanism and the architecture of the sites. We are using similar approaches to robe the structure, assembly, and topology of the enzyme. Studies of the topology of cytochrome b subunit using the phoA-fusion technique have supported our 8 membrane helic model, and we have done control experiments using R. sphaeroides reaction center L-sub-nit to demonstrate the validity of the phoa fusion technique We have completed the DNA-sequencing of the fbc operon coding for the three polypeptides (bis-cyt b, cyt cl and 2Fe.2S containing subunits) and derived the amino acid sequences. We have developed a preparative protocol to allow large-scale purification of a lightly active enzyme, and have completed an initial biochemical and biophysical charact- erization. Preliminary N-terminal sequencing of the subunits has confirmed the data derived from DNA-sequencing, and shown some post translational processing. By introducing a kanamycin cassette in place of the fbcf gene, we have prepared a strain lacking a functional fbc operon which was unable to grow photosynthetically (pho-). Complementation with the fbc operon on plasmid led to restoration of pho+ phenotype. We have made a number of site-directed mutants, and have started the detailed biophysical characterization of cinetic and thermodynamic parameters. We have also selected inhibitor resistant mutants, and made a preliminary characterization of myxothiazol resistant strains. We have constructed an apparatus for computer-aided fluorescence video imaging to aid in the screening and preliminary characterization of mutant strains. The software developed allows us to select strains with specific phenotypic attributes using false color coding of images, and measurement of induction kinetics of individual colonies.