GENETIC STUDIES OF HOW TURNS AFFECT PROTEIN STUCTURE

概述的研究目的是确定如何选择 不同的转角序列影响蛋白质的结构和稳定性。 这 工作将影响：（i）理解天然蛋白质如何折叠 （二）工程改造天然蛋白质变体的能力 具有改变的结构和期望的功能;以及（iii）能够 设计具有预定结构的全新大分子， 重要生物医学活动 为了确保这项工作的结果普遍适用， 将采用遗传方法来产生大量的 在两种不同的结构背景下有不同的话轮序列。 这两 结构背景将由α-螺旋蛋白表示， 细胞色素B-562和β折叠蛋白质质体蓝素。 具体 研究的目的是： 1)要创建每个特定转折处的不同序列库， 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回合序列;它们一起将代表 所有可能的氨基酸序列代替野生型转角序列。 2)为了在体内或在粗细胞裂解物中开发简单的颜色测定， 筛选文库中正确折叠的蛋白质。 3)为了确定这些代表性采样的转向序列， 图书馆. 通过将序列与颜色的结果进行比较， 屏幕上，将确定不同转动顺序的公差。 4)为了纯化和表征蛋白质，其中转角已被替换为 各种不同的氨基酸序列。 通过比较 的替代蛋白质，将有可能破译 一个回合的顺序和它在听写中的作用之间的关系 蛋白质结构和稳定性。

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tolerance for different turn sequences will be determined. 4) To purify and characterize proteins in which turns have been replaced by a variety of different amino acid sequences.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.