STRUCTURE/FUNCTION STUDIES OF HIV RNASE H

描述：(改编自申请人摘要)核糖核酸酶H(RNase H)是逆转录病毒生命周期的基本活动，例如 艾滋病毒，因此是一个重要的药物靶点。这样做的目的是 建议确定结构与功能的关系 在HIV-1逆转录酶的核糖核酸酶H区。这项研究 方法包括各种生物物理和遗传实验。这个 已知HIV逆转录酶的RNaseH结构域可折叠 变成一个类似RNaseH的结构基序，但复性的酶是 处于非活动状态。C末端不稳定和缺失的基本突起 已经被私家侦探推定在这一缺失中发挥重要作用 活跃度。为了获得HIV核糖核酸酶的选择性抑制剂，一种 活动隔离域是必需的。此域的活动变体将 由合理的设计和遗传选择的组合产生。 申请人已经获得了一个这样的依赖于Mn++的结构域 在初步结果中。这些活性物质的结构和稳定性 变异体将通过圆二色谱、荧光、核磁共振进行表征 和晶体结构研究。C-C的特殊作用- 终端区将进一步调查。初步研究显示 来自HIV这一区域的多肽是无结构的，而类似的 来自活性大肠杆菌同系物和MoLV的多肽被很好地折叠。 突变对分离结构域和多肽的影响将 被刻画出来。多肽抑制剂将被设计成与 蛋白质的这一区域。试图刻画和干扰 与结构/功能的关系，平衡和动力学 分离的RNaseH结构域的折叠特性也将是 调查过了。由于不对称的翻译后处理 HIV逆转录酶分子似乎需要一种核糖核酸酶 H结构域至少部分展开，这些研究也可能 了解这一机制。拟议研究的最终目的是 选择性地抑制这种基本的逆转录病毒活性。

DESCRIPTION: (Adapted from Applicant's Abstract) Ribonuclease H (RNase H) is an essential activity for the lifecycle of retroviruses such as HIV, and therefore represents an important drug target. The goal of this proposal is to determine the relationship between structure and function in the RNase H domain of HIV-1 reverse transcriptase. The research approaches include a variety of biophysical and genetic experiments. The isolated RNase H domain of HIV reverse transcriptase is known to fold into a RNase H-like structural motif, yet the refolded enzyme is inactive. Instability in the C-terminus and a missing basic protrusion have been postulated by the PI to play an important role in this lack of activity. In order to obtain selective inhibitors of HIV RNase, an active isolated domain is necessary. Active variants of this domain will be generated by a combination of rational design and genetic selections. One such Mn++-dependent domain has already been obtained by the applicant in preliminary results. The structure and stability of these active variants will be characterized by circular dichroism, fluorescence, NMR and crystallographic structural studies. The special role of the C- terminal region will be investigated further. Preliminary studies reveal that peptides from this region of HIV are unstructured, whereas similar peptides from the active E. coli homologue and MoLV are well folded. Effects of mutations on both the isolated domain and the peptides will be characterized. Peptide inhibitors will be designed that compete with this region of the protein. In an attempt to characterize and interfere with the structure/function relationship, the equilibrium and kinetic folding properties of the isolated RNase H domain will also be investigated. Since the asymmetric post-translational processing of the HIV reverse transcriptase molecule appears to require one of the RNase H domains to be at least partially unfolded, these studies may also shed light on this mechanism.The ultimate aim of the proposed research is selective inhibition of this essential retroviral activity.