REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

许多细胞质合成的蛋白质必须正确地靶向 非细胞质部位。出口蛋白质之间的统一特征 在所有的系统中都需要一个信号肽。校长 这项工作的目标是确定结构要求 蛋白质分泌所必需的信号肽和最终 本地化。然后，总体目标是确定为什么这些元素 很重要。为此，一系列系统的大肠杆菌突变体 将产生碱性磷酸酶基因。其中的每一个都将是 除了亚节段的氨基酸组成不同外，其他相同 氨基末端信号肽。在每个突变体中，这一区域将是 旨在测试构象、长度、疏水性和 总体拓扑。例如，我们现在有一系列突变体在 信号肽疏水核心区，含有均聚体和不同 系统地研究了核心区成分残基的疏水性 以及这一段的长度。这一方法将被开发为包括 一系列位于卵裂区的突变体旨在探索 传递到信号并被信号识别所需的结构特征 多肽酶。将构建一系列单独的更全球化的模型，以 探索子细分市场作为一个单位发挥作用的程度，并确定 拓扑对齐和方向的约束。The Rapid 开发每个包含多个残基取代的突变体将 通过开发适当的DNA限制酶切位点和 盒式诱变。这种方法便于比较整个 明确定义的平行结构中的结构单元。的能力 这些突变体支持碱性磷酸酶向E. 将通过活体研究对大肠杆菌周质进行评估。相对的 对电化学势的依赖，SecA和PrlA/Secy将是 已经成立了。运输缺陷突变体将进一步评估 在膜插入、转位、前体加工和 卵裂部位的保真度。这些研究旨在揭示 特定的信号序列在几个不同的步骤中至关重要 在运输过程中。分离的信号肽对应于 野生型和突变型序列将通过化学合成和 提纯以证实每种模型的物理特征。这些遗嘱 也可用于与可能具有以下特性的成分的结合研究，如SecA 参与运输过程中的特定步骤，目的是建立 体外结合的层次与我们在体内观察功能的层次相同。 增强正确率的信号肽的结构特征 在大肠杆菌中进行区段划分将有助于探测它们的真核细胞 对口单位。这些原则可以应用于组织特异性 治疗剂的靶向和运输工具的设计 其他蛋白质，包括真核蛋白质，进入大肠杆菌周质 用于随后的隔离。

Many cytoplasmically-synthesized proteins must be correctly targeted to noncytoplasmic locations. The unifying feature between exported proteins in all systems is the requirement for a signal peptide. The principal objective of this work is to determine the structural requirements of signal peptides that are necessary for protein secretion and final localization. The overall aim is then to determine why these elements are important. For this purpose, a systematic series of mutants of the E. coli alkaline phosphatase gene will be produced. Each of these will be identical except for the amino acid composition of a subsegment of the amino-terminal signal peptide. In each mutant, this region will be designed to test the role of conformation, length, hydrophobicity, and overall topology. For example, we now have a series of mutants in the signal peptide hydrophobic core region which contain homopolymers and vary systematically in the hydrophobicity of the core region contituent residue and the length of this segment. This approach will be developed to include a series of mutants in the cleavage region designed to explore the structural features required for delivery to and recognition by the signal peptidase. A separate series of more global models will be constructed to explore the extent to which subsegments function as a unit and to determine the constraints for topological alignment and orientation. The rapid development of mutants, each containing multiple residue substitutions will be accomplished by development of the appropriate DNA restriction sites and cassette mutagenesis. This approach facilitates the comparison of entire structural units in well-defined parallel constructions. The ability of these mutants to support the delivery of alkaline phosphatase to the E. coli periplasm will be evaluated through in vivo studies. The relative dependence on an electrochemical potential, SecA and PrlA/SecY will be established. Transport defective mutants will be further evaluated for competence in membrane insertion, translocation, precursor processing and fidelity of the cleavage site. These studies are designed to reveal the specific signal sequence features critical at several different steps during the transport process. Isolated signal peptides corresponding to the wild type and mutant sequences will be chemically synthesized and purified to substantiate the physical character of each model. These will also be used for binding studies with components, such as SecA, likely to be involved in a given step during transport, with the aim of establishing the same hierarchy for binding in vitro as we observe for function in vivo. The structural features of signal peptides which enhance correct compartmentalization in E. coli will be useful for probing their eukaryotic counterparts. These principles can be applied to the tissue-specific targeting of therapeutic agents and the design of vehicles to transport other proteins, including eukaryotic proteins, into the E. coli periplasm for subsequent isolation.