GENETIC STUDIES OF HOW TURNS AFFECT PROTEIN STUCTURE

轮次如何影响蛋白质结构的遗传学研究

• 批准号: 3468854
• 负责人: MICHAEL H HECHT
• 金额: $ 9.35万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468854
• 关键词: bioassay; calorimetry; chemical stability; circular dichroism; colorimetry; conformation; cytochrome b; globular protein; metalloproteins; molecular cloning; mutant; plant proteins; protein engineering; protein folding; protein purification; protein sequence; protein structure function; site directed mutagenesis; thermodynamics; tissue /cell culture; transfection; ultraviolet spectrometry

The objective of the research outlined is to determine how the choice of different turn sequences affects protein structure and stability. This work will impact upon: (i) the understanding of how natural proteins fold and function; (ii) the ability to engineer variants of natural proteins with altered structures and desired functions; and (iii) the capability to design entirely novel macromolecules with predetermined structures and biomedically important activities In order to insure that the results of this work are generally applicable, a genetic approach will be employed to generate a large collection of different turn sequences in two different structural contexts. These two structural contexts will be represented by the alpha-helical protein, cytochrome b-562 and the beta-sheet protein, plastocyanin. The specific aims of the research are: 1) To create a library of different sequences at specific turns in each of these two overexpressed model proteins. The members of the library will each contain a different turn sequence; and together they will represent all possible amino acid sequences in place of the wild-type turn sequence. 2) To develop simple color assays in vivo or in crude cell lysates to screen the libraries for correctly-folded proteins. 3) To determine the turn sequences for representative samplings of these libraries. By comparing the sequences with the results of the color screen, the tolerance for different turn sequences will be determined. 4) To purify and characterize proteins in which turns have been replaced by a variety of different amino acid sequences. By comparing the properties of the substituted proteins, it will be possible to decipher the relationship between the sequence of a turn, and its role in dictating protein structure and stability.

概述的研究的目标是确定如何选择 不同的转角序列会影响蛋白质的结构和稳定性。这 这项工作将影响：(I)对天然蛋白质如何折叠的理解 和功能；(Ii)改造天然蛋白质变体的能力 具有改变的结构和所需的功能；以及(Iii)能够 设计全新的具有预定结构的大分子和 重要的生物医学活动 为了确保这项工作的结果普遍适用， 将使用遗传方法来生成大量的 在两个不同的结构语境中的不同的转折顺序。这两个 结构背景将由α-螺旋蛋白代表， 细胞色素b-562和β-折叠蛋白，叶绿体蛋白。具体的 研究的目的是： 1)创建每个特定轮次的不同序列的文库 这两种过表达的模型蛋白。图书馆的成员将 每一个都包含一个不同的转弯顺序；它们将共同代表 所有可能的氨基酸序列取代野生型TURN序列。 2)在体内或在粗细胞裂解物中开发简单的显色分析以 筛选正确折叠的蛋白质文库。 3)确定这些具有代表性的样本的轮换顺序 图书馆。通过将序列与颜色的结果进行比较 屏幕上，将确定不同转弯顺序的公差。 4)纯化和鉴定转角被取代的蛋白质 各种不同的氨基酸序列。通过比较这些属性 在被取代的蛋白质中，将有可能破译 话轮顺序与其口述功能之间的关系 蛋白质的结构和稳定性。