REDESIGN OF STRUCTURAL REGIONS OF ALKALINE PHOSPHATASE

碱性磷酸酶结构区域的重新设计

• 批准号: 2178854
• 负责人: DEBRA A KENDALL
• 金额: $ 14.98万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178854
• 关键词: DNA; Escherichia coli; alkaline phosphatase; bacterial proteins; circular dichroism; conformation; gene expression; genetic manipulation; genetic models; hydropathy; membrane model; membrane permeability; membrane potentials; membrane proteins; mutant; nucleic acid sequence; peptide chemical synthesis; protein engineering; protein sequence; protein signal sequence; protein transport; site directed mutagenesis; structural genes

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.

许多细胞质合成的蛋白质必须正确靶向 非细胞质位置。 输出蛋白质之间的统一特征 在所有系统中，都需要信号肽。 校长 这项工作的目的是确定结构要求， 信号肽是蛋白质分泌和最终 本地化 总的目标是确定为什么这些元素是 重要. 为此目的，系统地研究了E.杆菌 将产生碱性磷酸酶基因。 每一个都将是 除了所述多肽的亚段的氨基酸组成之外， 氨基末端信号肽。 在每个突变体中，该区域将被 旨在测试构象，长度，疏水性和 整体拓扑 例如，我们现在有一系列的突变体， 信号肽疏水核心区，其含有均聚物且变化 系统地在核心区域组成残基的疏水性中 和这段的长度。 这一方法将包括 设计了一系列切割区突变体，以探索 信号传递和识别所需的结构特征 肽酶 将构建一系列单独的更具全球性的模型， 探索子段作为一个单元发挥作用的程度，并确定 拓扑对齐和方向的约束。 快速 突变体的开发，每个突变体含有多个残基取代， 通过开发适当的DNA限制性位点来实现， 盒式诱变 这种方法便于比较整个 在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. 将通过体内研究评价大肠杆菌周质。 的相对 根据电化学电势，SecA和PrIA/SecY将是 确立了习 将进一步评价转运缺陷突变体， 在膜插入，易位，前体加工和 切割位点的保真度。 这些研究旨在揭示 在几个不同步骤中关键的特定信号序列特征 在运输过程中。 分离的信号肽对应于 将化学合成野生型和突变体序列， 纯化以证实每个模型的物理特性。 这些将 也可用于与组分（如SecA）的结合研究， 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观察到的体内功能相同。 增强正确的信号肽的结构特征 E.大肠杆菌将有助于探测它们的真核生物 同行 这些原则可以应用于组织特异性 治疗剂的靶向和运输载体的设计 其他蛋白质，包括真核生物蛋白质，进入E.杆菌周质 随后隔离。