NMR DYNAMICS STUDY OF CHEMICAL ANALOGUES OF HIV-1 PROTEASE

HIV-1 蛋白酶化学类似物的核磁共振动力学研究

• 批准号: 7954643
• 负责人: STEPHEN B.H. KENT
• 金额: $ 0.47万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954643
• 关键词: Accounting; Aminoisobutyric Acids; Aspartate; Aspartic Endopeptidases; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Coupling; Distant; Enzymes; Equilibrium; Funding; Goals; Grant; HIV-1 protease; Hydrogen Bonding; Institution; Kinetics; Ligand Binding; Mechanics; Methods; Motion; Physiologic pulse; Property; Protein Dynamics; Proteins; Research; Research Personnel; Resources; Series; Source; Spin Labels; Surgical Flaps; Tertiary Protein Structure; Thermodynamics; United States National Institutes of Health; analog; base; chemical synthesis; dimer; enzyme activity; ionization

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Total chemical synthesis was used to prepare a series of unique chemical analogues of HIV-1 protease, where we systematically substituted the residues Gly51, Gly51' at the tips of the mobile 'flaps' (residues 37-61 in each domain of the protein homodimer) with L-Ala, D-Ala or Aib (alpha-aminoisobutyric acid) in both symmetric and asymmetric fashion. Such substitutions, although in regions distant from the catalytic aspartates, led in most cases to reduction of catalytic activity. In contrast to this, a 'covalent dimer' with L-Ala51 in one flap and D-Ala51' in another flap has shown native-like enzyme activity. We used continuous-wave and pulse EPR to characterize dynamic properties of flaps in spin-labeled analogues and found that the thermodynamic balance of the closed, semi-open and open ensembles (with respect to flaps) in HIV-1 protease analogues is perturbed in comparison to that of wild-type enzyme.We interpreted our results on the basis of Northrop's 'kinetic isomechanism' for aspartic proteases which employs a low-barrier hydrogen bond (LBHB) as the central part of the concept. We have developed Northrop's concept further to take into account dynamic properties of the protein and the non-equivalence of the flaps in the ligand-bound enzyme. We proposed a mechanical coupling of motions of the flaps and the catalytic residues Asp25 and Asp25'. On a mechanistic level, opening of the flaps corresponds to shortening of the distance between catalytic residues Asp25 and Asp25' thus promoting formation of the LBHB, and vice versa.Our goal is to employ NMR methods to detect and unambiguously prove presence of LBHB. In addition we would like to determine ionization states of catalytic residues for chemical analogues which demonstrate distinctly different protein dynamics and thus attempt to correlate dynamic properties of the enzyme with its chemical mechanism.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 用全化学合成方法合成了一系列独特的HIV-1蛋白酶类似物，系统地用L-丙氨酸、D-丙氨酸或α-氨基异丁酸(α-氨基异丁酸)以对称和不对称的方式取代了可移动的‘翻盖’(蛋白质均二聚体的每个结构域中的37-61个残基)顶端的Gly51，Gly51‘残基。这种取代虽然发生在远离催化天冬氨酸的区域，但在大多数情况下会导致催化活性的降低。与此形成鲜明对比的是，一种‘共价二聚体’--L-丙氨酸51和D-丙氨酸51‘在一种皮瓣中表现出天然的酶活性。我们使用连续波和脉冲EPR来表征自旋标记类似物中的翻盖的动态性质，发现HIV-1蛋白酶类似物中封闭、半开放和开放的系综(相对于翻盖)的热力学平衡与野生型酶相比被扰乱了。我们解释了我们的结果，这是基于诺斯罗普对天冬氨酸氨基转移酶采用低障碍氢键(LBHB)的动力学等机理。我们进一步发展了诺斯罗普的概念，以考虑蛋白质的动态性质和配体结合酶中翻盖的不等价性。我们提出了襟翼运动与催化残基Asp25和Asp25‘的机械耦合。在机理水平上，襟翼的打开对应于缩短催化残基Asp25和Asp25‘之间的距离，从而促进LBHB的形成，反之亦然。我们的目标是利用核磁共振方法来检测并明确证明LBHB的存在。此外，我们想要确定显示出明显不同的蛋白质动力学的化学类似物的催化残基的电离状态，从而试图将酶的动力学性质与其化学机制联系起来。