PARAMAGNETIC PROBES FOR TERTIARY STRUCTURE DETERMINATION

用于三级结构测定的顺磁探针

• 批准号: 2662359
• 负责人: THOMAS B WOOLF
• 金额: $ 10万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2662359
• 关键词: binding proteins; computer simulation; globular protein; lysozyme; membrane proteins; method development; molecular dynamics; nuclear magnetic resonance spectroscopy; protein structure; quantum chemistry; site directed mutagenesis

The development of a new method for tertiary structure determination of proteins is proposed. The approach may be applied to both membrane and globular proteins. As such, it represents a potentially powerful and novel method for new structure determination via a combination of NMR and computational methods. The approach uses cysteine scanning mutagenesis with site-directed paramagnetic probes to introduce paramagnetic centers in diamagnetic molecules. The resulting pseudocontact shifts are expressed as a function of the local tensor at the probe site and the distance and angle between the site and each nuclear spin. The nature of this new information: a) long-range, b) applied to multiple sites, and c) used in conjunction with other NMR information (e.g. NOEs, scalar coupling constants) provides restraints to be used in structure refinement. The approach will be tested in two systems with known tertiary structures. Computational methods will center on both ab initio calculations for the local g-tensor as well as the use of the CHARMm molecular dynamics program for refinement with pseudocontact shift restraints. The specific aims are: I A. Ab initio quantum chemical calculations of the g-tensor: The g- tensor, calculated via ab initio methods, will be used to determine the pseudocontact surface for structure refinement. A description of the change in tensor with molecular motion will be parameterized. I B. Molecular mechanics structure refinement. The restraints from pseudocontact induced shifts will be applied as energy penalties within the CHARMm code. Refinement will use simulated annealing. The accuracy of the approach will be tested with calculated dynamical trajectories. II A. Use of the g-tensor calculations for the zinc-finger binding protein. The cobalt substituted zinc-finger binding protein, with known tertiary structure, provides an initial test of the approach. II B. Site directed paramagnetic probes within T4-Lysozyme. Two probe types, optimized for shift effects and minimal relaxation effects, will be used. The T4-Lysozyme system will provide an excellent test of the approach for a globular protein with many mutants and known tertiary structures. The determination of new tertiary structures are important for rational drug design.

一种测定三级结构的新方法的建立 提出了蛋白质。 该方法可应用于膜和 球状蛋白质。 因此，它代表了一个潜在的强大和 通过核磁共振和核磁共振相结合确定新结构的新方法 计算方法。 该方法使用半胱氨酸扫描诱变 使用定点顺磁探针引入顺磁中心 在抗磁性分子中。 由此产生的赝接触位移是 表示为探针位置处的局部张量和 该位点与每个核自旋之间的距离和角度。 的性质 这一新信息：a) 远程，b) 适用于多个站点，以及 c) 与其他 NMR 信息（例如 NOE、标量 耦合常数）提供了结构中使用的约束 细化。 该方法将在两个已知的系统中进行测试 三级结构。 计算方法将集中于从头算起 局部 g 张量的计算以及 CHARMm 的使用 通过赝接触位移进行精化的分子动力学程序 限制。 具体目标是： I A. g 张量的从头算量子化学计算：g- 通过从头计算的张量将用于确定 用于结构细化的赝接触表面。 的描述 张量随分子运动的变化将被参数化。 I B. 分子力学结构细化。 的限制来自 赝接触引起的位移将作为能量惩罚应用 CHARMm 代码。 细化将使用模拟退火。 准确度 该方法的方法将通过计算的动态轨迹进行测试。 II A. 使用 g 张量计算来计算锌指结合 蛋白质。 钴取代的锌指结合蛋白，已知 三级结构，提供了该方法的初步测试。 II B. T4-溶菌酶内的定点顺磁探针。 两探头 针对转变效应和最小松弛效应进行优化的类型将是 用过的。 T4-溶菌酶系统将提供出色的测试 具有许多突变体和已知三级的球状蛋白的方法 结构。 新三级结构的确定对于理性研究非常重要 药物设计。