Aids Related Nmr Research

该项目利用核磁共振光谱学研究艾滋病毒和模型系统的分子组成部分。主要研究方向为：1）HIV逆转录酶核糖核酸酶H结构域的结构和动力学行为分析; 2）DNA pol β- a模型系统的动力学分析; 3）核苷酸对引物末端链的解封闭研究。我们最近确定了第一个解决方案的结构的15 kD RNA酶H结构域的HIV逆转录酶，使用[13 C，15 N]-标记的酶。该结构通常复制先前确定的晶体结构，除了C-末端螺旋（螺旋E）的共振受到极端交换增宽，排除了该元素的结构测定。我们还进行了15 N松弛研究RNase H域，以评估以前的建议，孤立的域是动态不稳定的，因此不活跃。在我们的研究条件下，我们发现RNase H结构域的动力学特性，即序参量、内扩散速率等与（活性）E. coli RNase HI酶，表明动态不稳定性可能不是导致分离结构域失活的原因。我们目前正在评估这个结构域与核苷酸和其他配体的相互作用，以更好地了解配体结合如何选择活性构象。在我们对碱基切除修复酶DNA pol β的研究中，我们现在已经分配了[甲基-13 C]甲硫氨酸标记酶中的所有甲硫氨酸甲基共振。目前，我们正在表征的构象和动态变化，导致从复合物的形成与缺口DNA和核苷三磷酸。M282甲硫氨酸共振的观察作为由核苷酸结合引起的大的开放->闭合构象转变的指示物是特别有用的，因为该残基位于经历大的构象变化的螺旋N上。最后，我们一直有兴趣了解AZTp 4A的命运，一个二核苷四磷酸，从AZT终止的引物由ATP的解封闭的结果。我们已经使用31 P NMR证明AZTp 4A核苷酸可以通过酶Ap 4A水解酶转化回AZT三磷酸（AZTTP）。后一种酶

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. The primary research areas are: 1) analysis of the structure and dynamic behavior of the Ribonuclease H domain of HIV reverse transcriptase; 2) dynamic analysis of DNA pol beta - a model system; 3) studies of the unblocking of primer-terminated strands by nucleotides. We recently determined the first solution structure of the 15 kD RNase H domain of HIV reverse transcriptase, using the [13C,15N]-labeled enzyme. The structure generally replicates the previously determined crystal structure, except that the resonances of the C-terminal helix (helix E) are subject to extreme exchange broadening, precluding a structural determination for this element. We have also performed 15N relaxation studies on the RNase H domain in order to evaluate a previous proposal that the isolated domain is dynamically unstable, and hence inactive. Under the conditions of our studies, we find that the dynamic characteristics, i.e. order parameters, internal diffusion rates, etc. of the RNase H domain are qualitatively similar to the (active) E. coli RNase HI enzyme, indicating that dynamic instability is probably not responsible for the inactivity of the isolated domain. We are currently evaluating the interaction of this domain with nucleotides and other ligands, in order to better understand how ligand binding selects the active conformation. In our studies of the base-excision repair enzyme DNA pol beta, we have now assigned all of the methionine methyl resonances in the [methyl-13C]methionine-labeled enzyme. We are currently characterizing the conformational and dynamic changes that result from complex formation with gapped DNA and nucleoside triphosphates. Observation of the M282 methionine resonance is particularly informative as an indicator of the large, open -> closed conformational transition which results from nucleotide binding, since this residue is located on helix N which undergoes a large conformational change. Finally, we have been interested in understanding the fate of AZTp4A, a dinucleoside tetraphoshate that results from the deblocking of AZT-terminated primers by ATP. We have used 31P NMR to demonstrate that the AZTp4A nucleotide can be converted back into AZT triphosphate (AZTTP) by the enzyme Ap4A hydrolase. The latter enzyme