The Functional Interplay of Lipid Membrane Components: Activation, Inhibition, and Raft Formation.

内向整流钾(KIR)通道和G蛋白偶联受体(GPCRs)是一种膜蛋白，它们是 受天然细胞膜中的胆固醇和阴离子脂类调节。我们将使用固态核磁共振 (SS核磁共振)研究双层环境中具有功能脂的蛋白质，范围从蛋白质脂质体到 生物膜。这些测量将补充功能分析、荧光技术和 相同条件下的分子动力学(MD)模拟。KIR通道与长QT综合征有关， 低血糖、巴特综合征、癫痫、药物滥用和周期性瘫痪。KIR通道是配体 但门控通道的结构和动力学的细节在很大程度上是未知的。Kir2通道系列 被阴离子脂质磷脂酰肌醇4，5-二磷酸(PIP2)门控，但被胆固醇灭活， 与PIP2竞争获取蛋白质。G蛋白激活的KIR通道(GIRK、Kir3)受共同作用的调控 PIP2和GBγ蛋白异源二聚体。在Kir3家族中，胆固醇增加而不是抑制活动。 在这里，我们将探索功能性脂质的不同作用，并量化观察到的结构和动力学 活动和非活动状态。我们将继续对KIR通道KirBac1.1的研究。我们分配了90%的 这种蛋白质中的15N和13C化学位移(超过1600个独特的重原子)，并利用这些赋值 鉴定变构，激活机制，结合胆固醇二聚体的失活结构，提炼 结构的关闭状态，并解决了结构的开启状态的通道。现在我们将测量 通道动态，并识别我们数据中反映的通道的多个门控状态。我们会研究 在电压作用下通道的结构变化，并使用 冻结捕获的动态核极化。同时，我们还将研究Kir3.1-KirBac1.3通道 奇美拉。初步数据确定了PIP2结合残基和膜-水界面残基 通道功能。最终的目标将是哺乳动物Kir3.2(GIRK2)通道及其完整的 功能性激活剂。在第二个项目中，我们将用CCL11研究CC基序趋化因子受体CCR3 脂双层中的趋化因子。没有针对CCR3的药物试验成功，这是不幸的，因为它参与了 癌症转移、HIV进入和COVID19细胞因子风暴。到目前为止，我们确定了CCL11对接和 信号转导依赖于双层胆固醇的剂量。SS核磁共振初步研究发现胆固醇 构象选择最佳的受体配基结合构型。我们计划全面分配15N CCR3在胆固醇和阴离子脂质富集膜中的13C化学位移。它的结构 将解决具有不同功能状态的CCLL11的蛋白质，并在随后测量区域动力学 为KirBac1.1建立了类似的工作流程。此外，还将使用核磁共振技术来解析CCL11在溶液中的结构 并与CCR3形成复合体。我们将推行胆固醇齐聚、CCR3二聚化及其与 在这些事件之间。在整个过程中，我们将研究脂质齐聚、动力学和蛋白质亲和力。

Inward-rectifier K+ (Kir) channels and G protein-coupled receptors (GPCRs) are membrane proteins that are regulated by cholesterol and anionic lipids found in their native membranes. We will use solid-state NMR (SSNMR) to study proteins with functional lipids in bilayer environments ranging from proteoliposomes to biological membranes. These measurements will compliment functional assays, fluorescence techniques, and molecular dynamics (MD) simulations under identical conditions. Kir channels are involved in long-QT syndrome, hypoglycemia, Bartter’s syndrome, epilepsy, substance abuse, and periodic paralysis. Kir Channels are ligand gated, but details of the structure and dynamics of gated channels are largely unknown. The Kir2 channel family is gated by the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) but inactivated by cholesterol which competes with PIP2 to access the protein. G protein-activated Kir channels (GIRK, Kir3) are gated by the coaction of PIP2 and Gbγ protein heterodimers. In the Kir3 family, cholesterol increases rather than suppresses activity. Here we will explore the differing roles of functional lipids and quantify the structure and dynamics of the observed active and inactivated states. We will continue our studies of the Kir channel, KirBac1.1. We assigned 90% of the 15N and 13C chemical shifts in this protein (over 1600 unique heavy atoms) and used these assignments to identify allostery, the activation mechanism, the inactivated structure bound to a cholesterol dimer, refined the structure of the closed state, and solved the structure of the open state of the channel. Now we will measure the channel dynamics and identify the multiple gated states of the channel reflected in our data. We will study structural changes in the channel under voltage and identify discrete channel states and lipid contacts using freeze-trapped Dynamic Nuclear Polarization. In tandem, we will also study the Kir3.1-KirBac1.3 channel chimera. Preliminary data identifies PIP2 binding residues and membrane-water interfacial residues key for channel function. The eventual goal will be the mammalian Kir3.2 (GIRK2) channel and its full complement of functional activators. In a second project we will study the CC motif chemokine receptor CCR3 with the CCL11 chemokine in lipid bilayers. No drug trial targeting CCR3 has succeeded, which is unfortunate as it is involved in cancer metastasis, HIV entry, and the COVID19 cytokine storm. To date, we identified both CCL11 docking, and signal transduction are dose dependent upon bilayer cholesterol. Preliminary SSNMR studies found cholesterol conformationally selects for optimal ligand binding configurations of the receptor. We plan to fully assign the 15N and 13C chemical shifts of CCR3 in cholesterol and anionic lipid enriched membranes. The structures of this protein with CCLL11 in different functional states will be solved, and regional dynamics measured following a similar workflow established for KirBac1.1. NMR will also be used to solve the structures of CCL11 in solution and in complex with CCR3. We will pursue cholesterol oligomerization, CCR3 dimerization, and the relationship between these events. Throughout we will examine lipid oligomerization, dynamics, and protein affinity.