NMR SOLUTION STRUCTURE OF COUPLING PROTEIN MMOB OF METHANE MONOOXYGENASE SYSTEM

我们计划解决MMOB的解决方案结构，一个小的16.5 kDa 一种调节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， 纯化方法，约18 mg纯的均质蛋白质的产量 每升M-9培养基中获得1000 μ g。 纯化的MMOB 蛋白质在25 ℃下是稳定的， 并冷冻干燥而没有显著的活性损失。 借助 我们记录了高分辨率的一维图像 和二维（NOESY和TOCSY）光谱，使用FBML 750 MHz 表明MMOB是NMR极好候选者 结构调查。 MMOB结构的确定 将涉及到使用homemandem进行顺序共振分配， (H)异源（H- 15 N）TOCSY和NOESY数据。 两个和三 三维TOCSY实验将用于鉴定氨基酸自旋 系统. CBCA（CO）NH中将使用统一标记的“C-“N-MMOB 和HNCACB研究，以完成伯氨基的分配 酸性序列 实验确定的距离和二面角 约束将用于分子动力学计算， 用模拟退火法求解MMOB结构。 高 现场750和600 MHz仪器在FBML和指导博士。 特纳在发展适当的多维 同性恋和异性恋实验是成功的关键 这次调查

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.