MOLECULAR ARCHITECTURE OF UQH2: CYT C2 OXIDOREDUCTASE

UQH 2：cyt c2的功能和分子结构 氧化还原酶正在通过一种综合的方法来研究球体 利用分子遗传学、生物化学和生物物理学。我们正在修改 编码酶的基因，并测定功能和 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 在导致抑制剂抗性的病变的残留水平上作图， 我们提出了一个8-跨膜的fetch模型，并定位了 两个醌反应性催化位点。我们已经开始修改残留物 可能在催化作用中起重要作用的位点上。我们的目标是 通过详细的生物物理方法， 通过位点特异性或随机诱变产生的突变体的研究，以及 利用这些信息来分析的机制和架构， 网站.我们正在使用类似的方法，以长袍的结构，大会， 和拓扑结构。细胞色素B的拓扑结构研究 亚基使用phoA融合技术支持我们的8膜 螺旋模型，并利用R. sphaeroides 反应中心L-sub-nit，以证明phoa融合的有效性 技术 我们已经完成了fbc操纵子编码的DNA测序， 三种多肽（含亚基的bis-cyt B、cyt cl和2Fe.2S） 并推导出氨基酸序列。我们开发了一种 允许大规模纯化轻度活性酶的方案， 并完成了初步的生物化学和生物物理特征- erization.亚基的初步N-末端测序 证实了来自DNA测序的数据，并显示了一些后 翻译加工通过将卡那霉素盒 我们制备了一种缺乏功能性fbc的菌株， 操纵子不能光合生长（pho-）。 与质粒上的fbc操纵子互补， pho+表型。我们已经制造了许多定点突变体， 开始详细的生物物理特性的动态和 热力学参数我们还选择了抗抑制剂 突变体，并对粘噻唑进行了初步表征 耐药菌株 我们已经建立了一个计算机辅助荧光视频装置 成像以帮助突变体的筛选和初步表征 菌株开发的软件使我们能够选择具有特定 使用图像的假彩色编码的表型属性和测量 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.