CONSTRUCTION OF NEW METALLOPROTEINS BY RATIONAL DESIGN

建议承担一系列项目的系统建设。 复制已知铜的最重要方面的铜中心 蛋白质中的化学，使用从头算合理蛋白质技术 设计。第一步是构建忠实的天然类似物 研究引入和控制所需因素的中心 蛋白质基质中的金属中心。第二步是系统地 改变设计中的金属几何形状，超出通常遇到的情况 以便探索结构/反应性关系。这个 蛋白质中新功能的构建需要详细的局部 相互作用是正确预测和形成的，总体来说， 保持了全球折叠和稳定。这项提议的目标是 专注于本地交互的设计。设计 全球折叠式IS的建议是专门专注于局部的设计 互动。全局折叠的设计被故意绕过 利用已知的蛋白质结构作为“支架”，在这种支架中， 通过大体上这样修改其序列来构建新的结合位点 新的配体结合位点是在不改变 整体脊椎折叠。一种新的分子建模技术应用于 这种“反向折叠”的方法是通过搜索已知的三种蛋白质 建立原生金属的突变的维度结构 协调球体和它解决了新的 站点和周围的蛋白质基质，同时保持原有的 脊椎折叠。设计是基于立体化学定义的 描述残数之间允许的几何关系的范围 和底物。鉴于它们相对几何上的简单性和 体积小，可提供多种X射线结构，范围广泛 光谱研究和丰富的模型工作，在 自然，铜矿为设计研究提供了有吸引力的目标，使用 这种新的“现场嫁接”方法。此外，他们的电子产品 结构，因此它们的反应性，是由能力控制的 蛋白质呈现一个刚性的配位体，在其中扰动金属 可以稳定地维护配位几何。静态分子 因此，建模技术特别适合于它们的 设计。铜或其他过渡金属的光谱研究可以 用来探测坐标球体的性质，以便 评估设计的成功并分析错误的性质 都是在失败中制造的。硫氧还蛋白，一种稳定的、单体的大肠杆菌蛋白 已知结构已被选为宿主蛋白，新的 中心将通过定点突变来构建。这个 金属场地的建造，以及协调的系统变化 用从头算方法研究蛋白质基质中的几何构型 蛋白质中的金属中心。最终，这将导致建设 具有在自然界中尚未遇到的反应性的地点，以及 生物技术应用的新型催化剂的设计。

It is proposed to undertake the systematic construction of a series of copper centers which reproduce the most important aspects of known copper chemistry in proteins, using techniques of de novo rational protein design. The first step is to construct faithful analogues of natural centers to study the factors that are required to introduce and control metal centers in a protein matrix. The second step is to systematically vary the metal geometry in the designs beyond that normally encountered in nature in order to explore structure/reactivity relationships. The construction of a new function in a protein requires that detailed local interactions are correctly predicted and formed, and that the overall, global fold and stability are maintained. The goal of this proposal is to focus specifically on the design of local interactions. The design a global fold is proposal is to focus specifically on the design of local interactions. The design a global fold is deliberately circumvented by using a known protein structure as a "scaffold" within which a completely new binding site is built by modifying its sequence substantially such that the new ligand binding site is accommodated without altering the overall backbone fold. A novel molecular modeling techniques applies this "inverse folding" approach by searching proteins of known-three dimensional structure for mutations that establish the primary metal coordination sphere and which resolve sterid conflicts between the new site and the surrounding protein matrix, while maintaining the original backbone fold. Designs are based on stereochemical definitions describing ranges of allowed geometrical relationships between residues and substrate. In view of their relative geometrical simplicity and small size, availability of several X-ray structures, extensive spectroscopic studies, and wealth of model work, important role in nature, copper sites present attractive targets for design studies using this new "site-grafting" approach. Furthermore, their electronic structure, and hence their reactivity, is controlled by the ability of the protein to present a rigid ligand sphere within which perturbed metal coordination geometries can be stably maintained. Static molecular modeling techniques are therefore particularly appropriate for their design. Spectroscopic studies of copper or other transition metals can be used to probe the nature of the coordination sphere in order to evaluate the success of the design and analyze the nature of the mistakes made in failures. Thioredoxin, a stable, monomeric E. coli protein of known structure has been chosen as the host protein into which the new centers will be constructed by site-directed mutagenesis. The construction of metal site, and systematic variation of coordination geometry within a protein matrix by de novo approach to the study of in metal centers in proteins. Ultimately this will lead to the construction of sites with reactivities not yet encountered in nature, and to the design of new catalysts for biotechnological applications.