Biophysical Properties Of Membranes Containing Polyunsaturated Phospholipids

含有多不饱和磷脂的膜的生物物理特性

• 批准号: 7591911
• 负责人: DRAKE C MITCHELL
• 金额: $ 35.1万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7591911
• 关键词: Area; Attention; Bos taurus; Cattle; Cholesterol; Chronic; Class; Differential Scanning Calorimetry; Diphenylhexatriene; Disease; Docosahexaenoic Acids; Equilibrium; Fluorescence Anisotropy; G Protein-Coupled Receptor Signaling; Goals; Kinetics; Lipids; Literature; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular; Neurons; Phospholipids; Process; Property; Proteins; Range; Rate; Relative (related person); Retinal; Rhodopsin; Sampling; Scanning; Solvents; Thermodynamics; Tissues; Transition Temperature; Variant; Vesicle; alcohol exposure; base; docosahexaenoylascorbic acid; interest; protein folding; reconstitution; relating to nervous system; rod outer segment disc; thermostability

An extensive literature, accumulated over the past forty years, demonstrates that variation of essentially any aspect of membrane lipid composition leads to modulation of the functional efficacy and/or oligomeric state of one or more membrane proteins. The effects that membrane composition may also have on membrane protein folding and structural stability have received somewhat less attention. Characterizing the fundamental forces that determine the thermodynamic stability of membrane proteins is an area of intense interest. The energetic contribution of lipid-protein interactions to membrane protein stability is likely to be as important for this class of proteins as the energetics of protein-solvent interactions is to the stability of soluble proteins. Determining the effects of membrane composition on the energy required for thermal denaturation provides a measure of the effects of membrane composition on relative protein folding energetics. Purified bovine rhodopsin was reconstituted into vesicles containing a range of cholesterol and docosahexaenoic acid (DHA, 22:6n3) acyl chains and rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate dependent, demonstrating the presence of a kinetically controlled component of denaturation. The activation energy of this kinetically controlled process (Ea) was determined from DSC thermograms by four separate methods. Both Tm and Ea varied with bilayer composition. The thermal stability of rhodopsin in SOPC reconstituted membranes was increased by the addition of 30 mol% cholesterol, and decreased by the addition of 20% SDPC. The simultaneous addition of cholesterol and SDPC further enhances the thermostability of rhodopsin beyond the effects of cholesterol alone, which may suggest the promotion of rhodopsin-cholesterol interactions by SDPC. Cholesterol increased Tm regardless of the level of DHA acyl chains. In contrast, cholesterol lowered Ea in the absence DHA acyl chains, but raised Ea in the presence of 20 mol% DHA-containing phospholipid. Relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. Tm for thermal unfolding decreased with increasing acyl chain disorder while kinetic stability (Ea) was maximal over a middle range of acyl chain order, such that highly ordered or disordered membranes resulted in kinetic destabilization. The activation energies of thermal denaturation in each of the samples, when considered as a function of acyl chain packing, demonstrate that the kinetic component of rhodopsin denaturation is maximally stabilized in membranes whose lipid packing is similar to that of the native disk membrane. These results establish that one important function of the combination of DHA acyl chains and cholesterol found in the rod outer segment disk membrane is to provide maximal kinetic stability for rhodopsin. The results also demonstrate that membrane composition has distinct effects on the thermal and kinetic stabilities of membrane proteins and suggest that a balance between membrane constituents that have opposite effects on acyl chain packing, such as DHA and cholesterol may be required for maximum protein stability.

在过去的四十年中积累的广泛文献表明，膜脂质组成的任何方面的变化都会导致一种或多种膜蛋白的功能功效和/或寡聚状态的调节。 膜成分也可能对膜蛋白折叠和结构稳定性产生的影响较少。 表征确定膜蛋白热力学稳定性的基本力是一个引起人们感兴趣的领域。 脂质 - 蛋白质相互作用对膜蛋白稳定性的能量贡献对于这类蛋白质可能与蛋白质 - 溶剂相互作用的能量学一样重要。 确定膜组成对热变性所需的能量的影响，可以衡量膜组成对相对蛋白质折叠能量的影响。 将纯化的牛动蛋白重构为含有一系列胆固醇和二十六六烯酸（DHA，22：6N3）酰基链的囊泡，并使用差分扫描量热法（DSC）检查了丙片链稳定性（DSC）。视紫红蛋白的热展开过渡温度（TM）取决于扫描速率，证明了具有动力学控制的变性成分。 通过四种单独的方法从DSC热合器确定该动力学控制过程（EA）的激活能。 TM和EA都随双层组成而变化。通过添加30 mol％胆固醇，增加了SOPC重建膜中视紫红质的热稳定性增加，并通过添加20％的SDPC降低。同时添加胆固醇和SDPC，进一步增强了视紫红质的热稳定性，超出了胆固醇的作用，这可能表明SDPC促进视紫红质 - 胆固醇的相互作用。无论DHA酰基链的水平如何，胆固醇会增加TM。 相比之下，胆固醇在不存在DHA酰基链的情况下降低了EA，但在20 mol％DHA的磷脂存在下升高了EA。 相对酰基链堆积顺序是从二苯基己二酰二酰化荧光各向异性衰减的测量结果确定的。用于热展开的TM随着酰基链障碍的增加而降低，而动力学稳定性（EA）在中间的酰基链顺序中最大，因此高度有序或无序的膜导致动力学不稳定。 在每个样品中，当将热变性的激活能视为酰基链堆积的函数时，表明在脂质填料与天然磁盘膜的膜相似的膜中，Rhodopsin denataturation的动力学成分最大地稳定。 这些结果表明，在棒外部段磁盘膜中发现的DHA酰基链和胆固醇组合的一个重要功能是为视紫红质提供最大的动力学稳定性。结果还表明，膜成分对膜蛋白的热和动力学稳定性具有明显的影响，并表明可能需要对酰基链填料具有相反作用的膜成分之间的平衡，例如DHA和胆固醇，以实现最大的蛋白质稳定性。