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首页> 外文学位 >Folding studies of outer surface protein A fragments from Borrellia burgdorferi: A model beta-sheet, and, Structural investigation of the catalytic doman of human type II inosine 5'-monophosphate dehydrogenase
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Folding studies of outer surface protein A fragments from Borrellia burgdorferi: A model beta-sheet, and, Structural investigation of the catalytic doman of human type II inosine 5'-monophosphate dehydrogenase

机译：伯氏疏螺旋体的外表面蛋白A片段的折叠研究：模型β-折叠，以及人类II型肌苷5'-单磷酸脱氢酶催化杜曼的结构研究

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摘要

The primary amino acid sequence of a protein dictates its overall three-dimensional structure, which arises through discrete folding processes. Outer Surface Protein A (OspA) represents a model system to investigate beta-sheet protein secondary structure formation. Through isolation of a unique single layer beta-sheet (SLB) segment from OspA that lacks tertiary contacts, the propensity for beta-sheet formation was examined in the absence of auxiliary interactions. A series of peptide segments from the SLB of OspA were investigated for conformation, thermodynamic stability, and mobility. These studies provided new insight into the importance of buried hydrophobic surface area and its influence on cooperative folding. Removal of the C-terminal domain of OspA allowed the unique SLB domain to be isolated and indicated this segment was competent to yield a fully folded protein. Analysis using Nuclear Magnetic Resonance Spectroscopy (NMR) generated structural and dynamic information for this N-terminal fragment of OspA and suggested that cooperative strand interactions and edge strand exposure significantly impacted folding. The study of OspA aids understanding of intra-molecular interactions that drive folding, specifically in the beta-sheet conformation.;The inter-molecular interactions that drive specific binding were investigated through Inosine 5'-Monophosphate Dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. As such, its inhibition leads to the depletion of guanine nucleotides, which disrupts DNA replication. Rapidly proliferating cells such as leukocytes and leukemic cells are particularly affected, thereby making IMPDH an attractive target for directed inhibition. Although the structure of IMPDH is known, better crystals diffracting to higher resolution should greatly improve existing knowledge of the enzyme active site. Here, a method is described for producing the catalytic domain of IMPDH, which was purified and crystallized for structural studies. Solubilization conditions were obtained by combining fusion-protein expression with sparse matrix screening, yielding a new high-throughput screening method relying on protease cleavage. The effects of chaotropic and molecular crowding agents on the structure of mobile regions of the protein significantly influenced the structure and activity of the enzyme. A new crystal structure refined to 2.45 A resolution reveals key active site amino acids interactions with substrate, co-factors and inhibitors and should lead to further therapeutic developments in IMPDH inhibitors that differentiate clinically relevant IMPDH isoforms. Both intra- and inter molecular interactions contribute to the final structure and function of proteins, the study of which enables better understanding of diseases, and through IMPDH has direct drug design applications.
机译：蛋白质的一级氨基酸序列决定了其整体三维结构，该结构是通过离散折叠过程产生的。外表面蛋白A（OspA）代表一个模型系统，用于研究β-折叠蛋白二级结构的形成。通过从缺乏三级接触的OspA中分离出唯一的单层β-折叠（SLB）片段，在没有辅助相互作用的情况下检查了形成β-折叠的倾向。研究了OspA SLB的一系列肽段的构象，热力学稳定性和迁移率。这些研究为掩埋疏水表面积的重要性及其对协同折叠的影响提供了新的见解。去除OspA的C-末端结构域使得可以分离独特的SLB结构域，并表明该区段能够产生完全折叠的蛋白质。使用核磁共振波谱（NMR）进行分析可得出OspA N端片段的结构和动态信息，并表明协作链相互作用和边缘链暴露会显着影响折叠。 OspA的研究有助于理解驱动折叠的分子内相互作用，特别是在β-折叠构象中。通过肌苷5'-单磷酸脱氢酶（IMPDH）研究了驱动特异性结合的分子间相互作用。 IMPDH是鸟嘌呤核苷酸从头合成中的限速酶。这样，其抑制作用导致鸟嘌呤核苷酸的消耗，这破坏了DNA复制。快速增殖的细胞如白细胞和白血病细胞受到特别影响，从而使IMPDH成为定向抑制的有吸引力的靶标。尽管IMPDH的结构是已知的，但衍射到更高分辨率的更好的晶体应大大改善酶活性位点的现有知识。在此，描述了一种用于生产IMPDH的催化结构域的方法，该方法经纯化和结晶用于结构研究。通过融合蛋白表达与稀疏基质筛选相结合获得增溶条件，从而产生了一种新的依靠蛋白酶裂解的高通量筛选方法。离液和分子拥挤剂对蛋白质可动区域结构的影响显着影响了酶的结构和活性。精制至2.45 A分辨率的新晶体结构揭示了关键的活性位点氨基酸与底物，辅因子和抑制剂的相互作用，并应导致IMPDH抑制剂的进一步治疗发展，这种抑制剂可区分临床相关的IMPDH同工型。分子间和分子间的相互作用都有助于蛋白质的最终结构和功能，对蛋白质的研究可以更好地了解疾病，并且通过IMPDH具有直接的药物设计应用。

著录项

  • 作者

    Gruswitz, Franz.;

  • 作者单位

    University of Rochester.;

  • 授予单位 University of Rochester.;
  • 学科 Biophysics.;Biophysics.
  • 学位 Ph.D.
  • 年度 2006
  • 页码 201 p.
  • 总页数 201
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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