NMR crystallography: Imaging active site chemistry and protonation states

我的团队正在致力于开发核磁共振辅助结晶学-- 固态核磁共振、X射线结晶学和计算化学-作为 酶活性部位的原子分辨探针，能够确定所有原子的位置，包括 氢气。通过定位氢原子，这项技术提供了通常关键的缺失的化学物质 链接结构和机制所需的信息，以及为Rational 治疗学的设计。这种方法有三个方面：使用X射线结晶学来提供粗略的结构 使用计算化学建立活性中心的化学详细模型的框架， 和探索的各种活性中心化学；这些模型可以通过比较来定量区分 他们预测的核磁共振化学位移与固体核磁共振实验的结果一致。提供了足够的 在活性中心内测量化学位移限制的数量，核磁共振辅助结晶学可以 唯一标识结构。目标系统包括依赖于吡哆醛-5‘-磷酸(PLP)的系统 酶，它与许多健康状况有关，并被用作治疗疾病的靶标，以及 β-内酰胺酶，介导抗生素对β-内酰胺类抗生素的耐药性。 PLP依赖的酶家族参与氨基酸和其他胺的代谢。 含有生物分子的。这种单一的辅因子可以参与一系列不同的化学转化， 包括外消旋、转氨基、α/β-脱羧基、α/β/γ-消除和取代。 了解活性中心如何针对这种不同的反应微调相同的辅因子是 这项提议。为了实现这一理解，核磁共振辅助结晶学被用来表征 这些具有原子分辨率的酶转化。在色氨酸合成酶中，这让我们可以凝视 该反应配位进出多个中间体。在这里，质子化状态完成了 例如，为什么像苯并咪唑这样的特定抑制剂不能反应形成 共价键，因为它通过氢键与βGlu109和带电的ε-氨基形成错误的方向 βLys87集团。 第二个目标是扩大在表征PLP依赖的酶转化方面的成功 酶对β-内酰胺酶，从TOHO-1β-内酰胺酶开始。在这里，我们以我们最初的化学位移为基础 用于研究抑制作用的化学机理的溶液动力学的指认和表征 抗生素。在这一应用中，将在与中子的界面上发展核磁共振辅助结晶学。 结晶学，到目前为止，它还不能在抑制剂存在的情况下解决结构，但在哪里 要在化学水平上理解这一机制，我们需要指定键的质子化状态 活性中心酸/碱催化残留物。

My group is working to develop NMR-assisted crystallography – the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry – as an atomic-resolution probe of enzyme active sites, capable of defining the position of all atoms, including hydrogens. By locating hydrogen atoms, this technique provides the often critical missing chemical information necessary to link structure and mechanism, as well as providing crucial information for the rational design of therapeutics. The approach is three-fold: X-ray crystallography is used to provide a coarse structural framework upon which chemically-detailed models of the active site are built using computational chemistry, and various active site chemistries explored; these models can be quantitatively distinguished by comparing their predicted NMR chemical shifts with the results from solid-state NMR experiments. Provided a sufficient number of chemical shift restraints are measured within the active site, NMR-assisted crystallography can uniquely identify the structure. The targeted systems include pyridoxal-5’-phosphate (PLP)-dependent enzymes, which have been implicated in numerous health conditions and as targets for treating diseases, and the β-Lactamases, which mediate antibiotic resistance to β-lactam antibiotics. The family of PLP-dependent enzymes are involved in the metabolism of amino acids and other amine- containing biomolecules. This single cofactor can participate in a diverse array of chemical transformations, including racemization, transamination, α/β-decarboxylation, and α/β/γ- elimination and substitution. Understanding how active sites fine-tune the same cofactor for such varied reactions is a primary objective of this proposal. To accomplish this understanding, NMR-assisted crystallography is employed to characterize these enzymatic transformations with atomic resolution. In tryptophan synthase, this allows us to peer along the reaction coordinates into and out of multiple intermediates. Here the protonation states complete the chemical picture for why, for example, specific inhibitors such as benzimidazole are unable to react to form a covalent bond as it is held in the wrong orientation by hydrogen bonds to βGlu109 and the charged ε-amino group of βLys87. A second goal is to extend the successes in characterizing enzymatic transformations in PLP-dependent enzymes to the β-lactamases, starting with the Toho-1 β-lactamase. Here we build on our initial chemical shift assignments and characterization of dynamics in solution to study the chemical mechanism used to inhibit antibiotics. In this application, NMR-assisted crystallography will be developed at the interface with neutron crystallography, which to date has been unable to solve the structure in the presence of an inhibitor, but where understanding the mechanism at the chemical level requires that we assign the protonation states of the key active site acid/base catalytic residues.