NMR SOLUTION STRUCTURE OF COUPLING PROTEIN MMOB OF METHANE MONOOXYGENASE SYSTEM

甲烷单加氧酶体系偶联蛋白MMOB的NMR溶液结构

• 批准号: 6279671
• 负责人: Stephen J. Lippard
• 金额: $ 0.59万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6279671
• 关键词: biological products; biomedical resource; enzymes

We plan to solve the solution structure of MMOB, a small 16.5 kDa enzyme that regulates electron and oxygen transfer reactions in the methane monooxygenase system. Using our current expression and purification methods, yields of -18 mg of pure, homogeneous protein per liter of M-9 culture medium are obtained. The purified MMOB protein is stable at 25 OC and can be concentrated in excess of 3 mM and freeze dried without significant loss of activity. With the help of Dr. Chris Turner, we have recorded high resolution one dimensional, and two dimensional (NOESY and TOCSY) spectra using the FBML 750 MHz spectrometer which suggest that MMOB is an excellent candidate for NMR structural investigation. The determination of the MMOB structure will involve making sequential resonance assignments using homonuclear (H) and heteronuclear (H- 15 N) TOCSY and NOESY data. Two and three dimensional TOCSY experiments will be used to identify amino acid spin systems. Uniformly labeled "C-"N - MMOB will be used in CBCA(CO)NH and HNCACB studies to complete the assignment of the primary amino acid sequence. Experimentally determined distance and dihedral angle constraints will be used in molecular dynamics calculations and simulated annealing refinement to solve the MMOB structure. The high field 750 and 600 MHz instruments at the FBML and guidance from Dr. Turner in the development of the appropriate multidimensional homonuclear and heteronuclear experiments are essential to the success of this investigation.

我们计划解决MMOB的溶液结构，一个小16.5 kDa 调节细胞内电子和氧转移反应的酶 甲烷单加氧酶系统。使用我们当前的表达式和 纯化方法，产率为-18 mg纯均一蛋白质 每升获得M-9培养基数。纯化的MMOB 蛋白质在25oC时稳定，可以浓缩到超过3 mM 冷冻干燥，活性无明显损失。在我的帮助下 克里斯·特纳博士，我们记录了一维的高分辨率， 和使用FBML 750 MHz的二维(NOESY和TOCSY)谱 这表明MMOB是核磁共振的一个很好的候选者 结构调查。MMOB结构的确定 将涉及到使用同核进行顺序共振分配 (H)和异核(H-15N)TOCSY和NOESY数据。二和三 将使用三维TOCSY实验来识别氨基酸自旋 系统。统一标记的“C-”N-MMOB将用于CBCA(CO)NH 和HNCACB研究完成伯氨基的指定 酸序列。实验确定的距离和二面角 约束将用于分子动力学计算和 模拟退火法求精化MMOB结构。高潮 FBML的现场750和600 MHz仪器以及Dr。 特纳在发展中的适当的多维 同核和异核实验是成功的关键 这起调查的责任。