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Understanding the relationship between sequence and structure: Biophysical studies of beta-bundles and bipartite tetracysteine display in beta sheets.

机译:了解序列和结构之间的关系:β束和二分体四半胱氨酸在β折叠中的生物物理研究。

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摘要

1. Biophysical studies of beta-bundles. Chapter 1 provides an overview of the work on beta-peptide assembly (oligomers of beta3-amino acids) into quaternary structures known as bundles. In particular, the review highlights the work of the Schepartz laboratory, and the progress made thus far towards the rational design of novel non-proteinaceous beta-peptide assemblies with stabilities, and eventually, functionalities analogous to those of natural proteins.1-4.;Chapter 2 details the extensive biophysical characterization (analytical ultracentrifugation (AU), circular dichroism (CD), 1-anilino-8-naphthalenesulfonate (ANS) binding and deuterium exchange NMR (DX NMR)) of an octamer beta-peptide bundle, Acid-1Y. Surprising, we found that Acid-1Y is more stable than its beta-peptide predecessors, Zwit-1F and Acid-1F/Base-1F. Following on this work, the x-ray structural characterization of Zwit-YK, the most stable bundle to date, is described.;Chapter 3 investigates whether beta3-peptide bundle stoichiometry is controlled by the side chain identity within the bundle core. Specifically, we investigated mutations of leucine to valine in the sequences of the octameric bundles. These second generation valine beta-peptides assemble into discrete and stable tetrameric bundles that were extensively characterized by AU, CD, ANS binding and DX NMR.13.;Our interest in modifying the core of beta-bundles was extended in Chapters 4 and 5. These chapters detail the biophysical characterization of novel beta-bundles with alternative core sequences that were predicted using Rosetta (in collaboration with Professor Rhiju Das, Stanford University and Professor David Baker, University of Washington).;Finally, the evolution of bundles with functions that mimic those of natural proteins requires the advancement of screening techniques that will quickly identify beta-peptide sequences that assemble into bundles. Chapter 6 overviews two methods that enable efficient and facile screening of beta-peptide bundle assembly.;2. Bipartite tetracysteine display in beta sheets. The Schepartz laboratory has shown the utility of terminal split-tetracysteine motifs in detecting protein oligomerization and distinguishing between folded and unfolded proteins. Chapter 7 describes the extension of this work in which we engineered intramolecular split tetra-cysteine motifs. We found that this method favors sites with flexible geometries such as loops. This information is critical for directing successful bipartite tetracysteine site designs.
机译:1.β-束的生物物理研究。第1章概述了将β肽(β3氨基酸的寡聚体)组装成称为束的四级结构的工作。特别是,这篇综述着重介绍了Schepartz实验室的工作,以及迄今为止在合理设计具有稳定性,并最终类似于天然蛋白的功能的新型非蛋白质β-肽装配体方面取得的进展。1-4。 ;第二章详细介绍了八聚体β肽束酸的广泛生物物理特征(分析超速离心(AU),圆二色性(CD),1-苯胺基-8-萘磺酸盐(ANS)结合和氘交换NMR(DX NMR)) -1Y。令人惊讶的是,我们发现Acid-1Y比其前任肽Zwit-1F和Acid-1F / Base-1F更稳定。继这项工作之后,描述了迄今为止最稳定的束Zwit-YK的X射线结构表征。;第3章研究了β3肽束化学计量是否受束核内的侧链同一性控制。具体来说,我们调查了八聚体束序列中亮氨酸到缬氨酸的突变。这些第二代缬氨酸β肽组装成离散且稳定的四聚体束,其特征在于AU,CD,ANS结合和DX NMR(13);我们在第4章和第5章中扩展了对β束核心的修饰兴趣。这些章节详细介绍了使用Rosetta(与斯坦福大学的Rhiju Das教授和华盛顿大学的David Baker教授合作)预测的具有可选核心序列的新型β-束的生物物理特性;最后,具有功能的束的进化模仿天然蛋白质的蛋白质需要先进的筛选技术,该技术将快速识别组装成束的β肽序列。第6章概述了两种方法,它们可以高效,轻松地筛查β-肽束组装。2。双半胱氨酸在β片中展示。 Schepartz实验室显示出末端四分体半胱氨酸基序在检测蛋白质寡聚化以及区分折叠和未折叠蛋白质方面的实用性。第7章描述了这项工作的扩展,在其中我们设计了分子内分裂的四半胱氨酸基序。我们发现,这种方法有利于具有灵活几何形状(例如回路)的站点。该信息对于指导成功的二分体半胱氨酸位点设计至关重要。

著录项

  • 作者

    Goodman, Jessica Leigh.;

  • 作者单位

    Yale University.;

  • 授予单位 Yale University.;
  • 学科 Biophysics General.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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