NMR Investigations Of Cell Membrane Structure

我们在大肠杆菌中重组表达CB 2作为与麦芽糖结合蛋白和几个亲和标签的融合物。将CB 2融合蛋白溶解、纯化、切割融合物，并从切割产物中再次纯化CB 2。我们广泛研究了去污剂、脂质和大麻素配体对重组大麻素受体CB 2稳定性的影响。这一努力产生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）和高亲和力大麻素配体CP-55，940或SR-144，528的协同作用是有效稳定十二烷基麦芽糖苷（DDM）/ CHAPS洗涤剂溶液中CB 2的功能折叠所需的。与CHS类似，带负电荷的丝氨酸头基（PS）磷脂在胶束中发挥了显着的稳定作用，而不带电荷的磷脂是无效的。 纯化的CB 2重构成脂质双层保留功能长达数周，使得能够在生理相关条件下对该GPCR进行高分辨率结构研究。 已经实现了以毫克水平重构功能性CB 2，并浓缩至40微升的体积，足以通过固态NMR进行结构研究。通过使用放射性配体以及氘代配体结合2 H-MAS NMR的配体结合以及通过在GTP γ S放射性测定中使用重组产生的G蛋白的G蛋白活化研究来验证受体的功能性。 通过分析NMR、使用标记脂质和CB 2的荧光光谱、动态光散射和蔗糖梯度离心研究了脂蛋白体的组成、大小和均匀性。探索性的NMR实验上进行的2毫克样品的均匀13 C-和15 N标记的CB 2和比较的实验结果与模拟光谱从原子坐标的CB 2模型已证明的实验概念的可行性。 具体的同位素标记方案正在开发中，以实现结构分析所需的光谱分辨率。 CB 2的结构和功能研究可能受益于将纯化的功能性受体以受控的密度固定到合适的表面上，并且优选地以均匀的取向固定。我们开发的策略，制备功能性重组CB 2和固定在固体界面。通过表面等离子体共振证实了CB 2的成功沉积。 具有高含量的多不饱和磷脂酰乙醇胺（PE）的膜促进后视紫红质-II（MII）的形成，后视紫红质-II是激活G蛋白转导的牛视紫红质的光中间体。我们确定MII形成是否与PE的弹性性能定量相关。本文研究了反六方相多不饱和脂18：0-22：6 n-3 PE、18：0-22：5 n-6PE和模型脂18：1 n-9-18：1 n-9 PE的单分子膜的曲率弹性。所有三种脂质形成具有26-28埃的相当低的自发曲率半径的脂质单层。含有高浓度多不饱和PE的膜中的负曲率弹性应力非常高。 从受体周围的脂质层释放这种压力的一小部分就足以使MI/MII平衡向MII转移，MII是激活G蛋白的状态。此外，多不饱和双层具有约27 A的疏水厚度，其已被确定为匹配视紫红质的疏水跨膜螺旋的长度。数据表明，多不饱和脂质是重要的A类GPCR激活，我们推测，视紫红质模型是特别相关的组成活性的GPCR和激活弱激动剂。 与MPBS合作，NINDS通过进行固态NMR实验对电压门控钾通道KvAP进行了研究。我们对通道S1-S4电压传感结构域的实验表明，脂质与蛋白质具有微弱且短暂的相互作用，不会明显改变该结构域的结构。在脂质膜中嵌入的S1-S4结构域上获得的数据与在洗涤剂胶束中获得的结构域的X射线和溶液NMR结构非常一致。S1 S4结构域表现出与脂质的广泛相互作用，并且当嵌入膜中时，该结构域严重水合。有证据表明阴离子脂质与S1 S4结构域的一些优先相互作用。在电压传感中起主要作用的S1 S4结构域内的精氨酸残基被充分水合，并且被定位在非常接近脂质的位置，表现出与脂质头基和酰基链的密切相互作用。 我们继续研究脂质基质的生物物理性质，这些性质对膜蛋白的功能很重要。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。胆固醇和两种脂质的混合物的液体有序相被证明本身是不均匀的。横向偏析内相观察，与六方填充的饱和链的区域分离的间隙区域富含胆固醇和不饱和链。观察到的子结构解释了现有的实验数据，并提供了一个重点，为未来的努力，旨在了解细胞膜的分子尺度结构。这张相的图片为许多实验结果提供了解释，其中大部分是通过NMR获得的，这些结果到目前为止还缺乏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 high resolution 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. Exploratory NMR experiments conducted on a 2-mg sample of homogeneously 13C- and 15N labeled CB2 and comparison of experimental results with simulated spectra obtained from the atomic coordinates of a CB2 model have demonstrated feasibility of the experimental concept. Specific isotopic labeling schemes are under development to achieve the desired spectral resolution for structural analysis. 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. Membranes with a high content of polyunsaturated phosphatidylethanolamines (PE) facilitate formation of metarhodopsin-II (MII), the photointermediate of bovine rhodopsin that activates the G protein transducin. We determined whether MII-formation is quantitatively linked to the elastic properties of PEs. Curvature elasticity of monolayers of the polyunsaturated lipids 18:0-22:6n-3PE, 18:0-22:5n-6PE and the model lipid 18:1n-9-18:1n-9PE were investigated in the inverse hexagonal phase. All three lipids form lipid monolayers with rather low spontaneous radii of curvature of 26-28 Angstrom. Negative curvature elastic stress in membranes containing high concentrations of polyunsaturated PEs is very high. Release of even a small fraction of this stress from the layer of lipids surrounding the receptor is sufficient to shift the MI/MII equilibrium towards MII, the state that activates G protein. Furthermore, polyunsaturated bilayers have a hydrophobic thickness of about 27 A which has been determined to match the length of the hydrophobic transmembrane helices of rhodopsin. The data show that polyunsaturated lipids are important for class A GPCR activation, and we speculate that the rhodopsin model is particularly relevant for constitutive activity of GPCR and activation by weak agonists. In collaboration with MPBS, NINDS we contribute to investigation of the voltage-gated potassium channel KvAP by conducting solid-state NMR experiments. Our experiments with the S1-S4 voltage sensing domain of the channel suggest that lipids have weak and transient interactions with the protein that do not detectably alter the structure of the domain. Data obtained on S1-S4 domain embedded in lipid membranes are remarkably consistent with both X-ray and solution NMR structures of the domain obtained in detergent micelles. The S1S4 domain exhibits extensive interactions with lipids and the domain is heavily hydrated when embedded in a membrane. There is evidence for some preferential interactions of anionic lipids with the S1S4 domain. The arginine residues within the S1S4 domain, which play a major role in voltage sensing, are well hydrated and are positioned in close proximity to lipids, exhibiting close interactions with both lipid headgroups and acyl chains. 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.