NMR Investigations Of Cell Membrane Structure

我们在大肠杆菌中以麦芽糖结合蛋白和几个亲和标签的融合形式重组表达了CB2。CB2-融合蛋白被溶解、纯化、融合裂解，并从裂解产物中再次纯化CB2。我们广泛研究了洗涤剂、脂类和大麻素配体对重组大麻素受体CB2稳定性的影响。这项工作导致了适用于广泛下游应用的全功能受体的制备和处理指南。我们证明了阴离子胆固醇衍生物胆固醇半琥珀酸酯(CHS)和高亲和力的大麻素配体CP-55,940或SR-144,528的协同作用是有效稳定CB2在十二烷基麦芽糖苷(DDM)/CHAPS洗涤剂溶液中功能折叠所必需的。与CHS类似，带有丝氨酸头基的带负电荷的磷脂在胶束中具有显著的稳定作用，而不带电荷的磷脂则不起作用。重组成脂质双层的纯化的CB2保留了长达几周的功能，从而能够在生理相关的条件下对这种GPCR进行结构研究。 已经实现了功能CB2在毫克水平上的重组，并浓缩到40微升的体积，足以用于固体核磁共振的结构研究。结合~2H-MAS核磁共振和重组产生的G蛋白在GTP-GammaS放射分析中的G蛋白活化研究，用放射性配体和氚配体结合来验证该受体的功能。用分析核磁共振、标记脂质和CB2荧光光谱、动态光散射和蔗糖梯度离心法研究了蛋白质脂质体的组成、大小和均一性。通过氨基酸的化学标记以及氨基酸的特定同位素标记的特定同位素标记方案正在开发中，以实现所需的光谱分辨率，以便通过核磁共振和电子顺磁共振进行结构分析。为了缩小具有珍贵的同位素标记GPCR的样品的大小，我们合作将动态核极化(DNP)技术应用到我们的样品中，将灵敏度提高了一到两个数量级。这些研究的目标是确定作为与受体结合的配体的功能的结构差异。 GPCR二聚化是细胞信号通路中调节GPCRs生物合成、成熟、配体结合、与G蛋白偶联及下游信号转导的重要机制。然而，确定膜中GPCR的寡聚状态是具有挑战性的。探讨了小角中子散射(SANS)是否适合研究受体功能状态下的GPCR齐聚状态。实验是用质子化的牛视紫红质重组成过氚化脂基质进行的。这在脂类和蛋白质之间产生了最大的中子散射长度密度对比。通过在过氚缓冲液中进行实验，将中子的非相干散射降到最低。观察到，当脂质与蛋白质摩尔比接近500/1时，脂质基质中视紫质齐聚的状态取决于受体的光激活状态。虽然暗适应视紫红质是单体，但漂白的视紫红质主要形成二聚体。 CB2的结构和功能研究可能受益于将纯化的和功能的受体以可控的密度固定在合适的表面上，最好是以均匀的方向固定。我们开发了制备功能性、重组CB2和固相界面固定化的策略。表面等离子体共振谱证实了CB2的成功沉积。 我们与爱尔兰都柏林三一学院马丁·卡弗里实验室合作，研究了脂蛋白信号肽II(LspA)的结构，LspA是铜绿假单胞菌的关键酶。其功能是从前脂蛋白中释放信号肽。该项目的目标是收集与LspA抑制和脂蛋白处理相关的结构信息。对LspA在洗涤剂胶束中进行了溶液-状态核磁共振实验，并对重组为单油酸立方相的LspA进行了固态核磁共振实验。获得了LspA的载脂蛋白形式和球霉素结合型的光谱。 我们继续对脂质基质的生物物理性质进行研究，这些性质对于完整的膜蛋白的功能是重要的。与进行分子模拟的实验室合作，我们探索了在膜中存在高胆固醇浓度时形成的液体有序相的内部结构。胆固醇和两种脂类混合物的液体有序相本身是不均匀的。在相中观察到横向偏析，六角堆积的饱和链区域被富含胆固醇的间隙区域和不饱和链隔开。观察到的亚结构解释了现有的实验数据，并为未来旨在了解细胞膜分子尺度结构的努力提供了重点。这张相图为许多实验结果提供了解释，其中大多数实验结果是由核磁共振获得的，到目前为止，这些实验结果缺乏对分子模型的一致描述。

We express CB2 recombinantly in Escherichia coli as a fusion with maltose-binding protein and several affinity tags. The CB2-fusion protein is solubilized, purified, the fusion cleaved, and CB2 purified again from cleavage products. We extensively studied the effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2. The effort resulted in guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/ CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling structural studies of this GPCR at physiologically relevant conditions. Reconstitution of functional CB2 at the level of milligrams, and concentration to a volume of 40 microliters, sufficient for structural studies by solid state NMR has been achieved. Functionality of the receptor was verified by ligand binding using radioactive ligands as well as deuterated ligands in combination with 2H-MAS NMR and by G protein activation studies using recombinantly produced G protein in a GTPgammaS radioactive assay. Composition, size, and homogeneity of proteoliposomes were investigated by analytical NMR, fluorescence spectroscopy using labeled lipid and CB2, dynamic light scattering, and sucrose gradient centrifugation. Specific isotopic labeling schemes by chemical labeling of amino acids as well by specific isotopic labeling of amino acids are under development to achieve the desired spectral resolution for structural analysis by NMR as well as EPR. To reduce the size of samples with precious isotopically labeled GPCR, we collaborate on application of dynamic nuclear polarization (DNP) techniques to our samples that raise sensitivity by one to two orders of magnitude. The goal of these studies is to determine structural differences as a function of ligands that are bound to the receptor. Dimerization of GPCR has emerged as an essential mechanism regulating GPCR biosynthesis, maturation, ligand binding, coupling with G protein and downstream signaling in cell-signaling pathways. However, determining the oligomeric state of a GPCR in a membrane is challenging. It was explored if small angle neutron scattering (SANS) is a suitable tool to study the state of GPCR oligomerization at functional conditions of the receptor. Experiments were conducted with protonated bovine rhodopsin reconstituted into a perdeuterated lipid matrix. This yields maximal neutron scattering length density contrast between lipid and protein. Incoherent scattering of neutrons was minimized by conducting experiments in perdeuterated buffer. It was observed that the state of rhodopsin oligomerization in a lipid matrix at lipid-to-protein molar ratios near 500/1 depends on the state of photoactivation of the receptor. While dark-adapted rhodopsin was monomeric, bleached rhodopsin formed mostly dimers. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. We develop strategies for preparation of functional, recombinant CB2 and immobilization at solid interfaces. The successful deposition of CB2 was demonstrated by surface plasmon resonance. In collaboration with the Laboratory of Martin Caffrey, Trinity College, Dublin, Ireland, we studying the structure of lipoprotein signal peptidase II (LspA), a membrane protein which is a key enzyme in Pseudomonas aeruginosa. Its function is to release signal peptides from prolipoproteins. The goal of the project is to gather structural information related to LspA inhibition and lipoprotein processing. Both solution-state NMR experiments on LspA in detergent micelles and solid-state NMR experiments on LspA reconstituted into a monoolein cubic phase are conducted. Spectra of both the apo form of LspA as well as the globomycin-bound form were obtained. We continue our studies on biophysical properties of the lipid matrix that are important for function of integral membrane proteins. In collaboration with laboratories that conduct molecular simulations, we explored the internal structure of the liquid ordered phase that forms in the presence of high cholesterol concentrations in membranes. The liquid ordered phase of a mixture of cholesterol and two lipids was shown to be itself inhomogeneous. Lateral segregation within the phase is observed, with regions of hexagonally packed saturated chains separated by interstitial regions enriched in cholesterol and unsaturated chains. The observed substructure explains existing experimental data and provides a focus for future efforts aimed at understanding the molecular scale structure of cell membranes. This picture of the phase provides an explanation for a number of experimental results, most of them obtained by NMR, which have until now lacked a consistent description in terms of a molecular model.