REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

碱性磷酸酶信号肽介导细胞转运 将该酶转移到大肠杆菌周质中。我们将研究 促进出口的多肽的结构特征 进程。将产生与野生的完全相同的突变体 类型碱性磷酸酶，但在氨基酸序列中除外 信号肽核心区。对于每个突变体，这一区域将 含有一种确定的氨基酸组成，最适合 一种特殊结构元素的形成。这些结构 单元的设计将测试构象、长度和 信号肽功能中的疏水性。快速发展 每个都包含多个残基替换的突变体将是 通过在两侧创建独特的限制点来实现 核心区。通过替换将生成新的信号序列 人工合成寡核苷酸编码的野生型DA序列 用于待测试的结构模型的氨基酸序列。 这些突变体支持分泌过程的能力将 通过体内转运研究进行评估。为了进一步 区分二级结构和一级序列的作用 信号肽功能的特异性，其他突变体将是 其包含来自信号序列的核心区 其他分泌的蛋白质。每个突变体的结构特征 序列将通过分析 相应的化学合成信号肽。同舟共济 这些研究将阐明两者之间的相互关系 每种信号肽的结构性质和体内功能。 关于构象进化的信息 补体膜相互作用与细菌的互换性 这些结构单元随后将应用于重新设计 成熟蛋白质。成熟蛋白质的区域将被取代 具有新的结构部分，旨在确定 膜的稳定和转化的要求 碱性磷酸酶转化为跨膜蛋白的预测 定位。这一方法将阐明以下准则 从总体上重新设计蛋白质的结构域。这个 增强蛋白质的信号肽的结构特征 换乘可以应用于交通工具的设计 其他蛋白质，包括真核蛋白质，进入大肠杆菌 用于后续分离的周质。

The signal peptide of alkaline phosphatase mediates transport of the enzyme to the E. coli periplasm. We will examine the structural features of this peptide which facilitate the export process. Mutants will be produced which are identical to wild type alkaline phosphatase except in the amino acid sequence of the signal peptide core region. For each mutant, this region will contain a defined amino acid composition which is optimal for formation of a particular structural element. These structural units will be designed to test the role of conformation, length, and hydrophobicity in signal peptide function. The rapid development of mutants, each containing multiple residue substitutions, will be accomplished by creating unique restriction sites flanking the core region. New signal sequences will be generated by replacing the wild type DA sequence with synthetic oligonucleotides coding for the amino acid sequence of the structural model to be tested. The ability of these mutants to support the secretion process will be evaluated through in vivo transport studies. To further distinguish the role of secondary structure and primary sequence specificity in signal peptide function, additional mutants will be made which contain the core region from signal sequences of other secreted proteins. The structural character of each mutant sequence will be substantiated through analysis of the corresponding chemically-synthesized signal peptides. Together these studies will elucidate the interrelationship between the structural properties of each signal peptide and in vivo function. The information which evolves regarding conformations which complement membrane interactions and the interchangeability of these structural units will then be applied to the redesign of the mature protein. Regions of the mature protein will be replaced with new structural segments designed to determine the requirements for membrane stabilization and transformation of alkaline phosphatase into a transmembrane protein with predicted orientation. This approach will elucidate the guidelines for redesigning structural domains in proteins in general. The structural features of signal peptides which enhance protein translocation can be applied to the design of vehicles to transport other proteins, including eukaryotic proteins, into the E. coli periplasm for subsequent isolation.