HIGH DENSITY LIPOROTEIN STRUCTURE-FUNCTION CORRELATIONS

本研究的长期目标是阐明高密度脂蛋白（HDL）的结构-功能关系。在循环中，HDL溶解脂质，从外周细胞中清除胆固醇，激活胆固醇的酯化反应，并将胆固醇酯输送到肝脏和甾体生成组织进行代谢和排泄。HDL的这些功能是由其主要蛋白质成分载脂蛋白A-I （apoA-I）介导的，是这类脂蛋白抗动脉粥样硬化作用的基础。在HDL代谢的几个关键步骤中，apoA-I发生构象变化以适应HDL脂质含量的变化，使apoA-II与HDL结合，并可能介导与膜和卵磷脂胆固醇酰基转移酶（LCAT）的相互作用。一个假定的“铰链”区域与apoA-I的构象变化有关。该项目的具体目标是确定apoA-I负责“铰链”功能的螺旋结构和调节“铰链”结构域构象适应性的关键氨基酸，并研究apoA-I的“封闭”和“开放”铰链形式之间的结构和动态差异。为了确定参与apoA-I“铰链”功能的螺旋，我们提出在大肠杆菌中构建和表达apoA-I突变体，其中每个候选螺旋都被apoA-I的第一个或最后一个螺旋取代，这些螺旋与脂质紧密结合且不移动。通过光谱学（CD和荧光）方法对突变体进行结构研究并研究其脂质结合特性后，将对定义的RHDL颗粒中的apoa - 1突变体的“hinge”功能进行检查：颗粒重排、apoa - 2结合、与双层膜的相互作用和LCAT激活。通过构建apoA-I的Cys突变体作为特定交联或荧光标记的位点，将确认“铰链”螺旋的身份，并研究结构重排及其动力学。疏水性、电荷分布和Pro残基在“铰链”螺旋（es）中的作用将通过单个氨基酸的诱变取代来评估。这些研究有望定位apoA-I的“铰链”区域，并阐明其在apoA-I的几个重要功能中的作用机制。

The long-term goal of this research is to elucidate the structure-function relationships in high density lipoproteins (HDL). In circulation, HDL solubilizes lipids, removes cholesterol from peripheral cells, activates the esterification of cholesterol, and delivers cholesterol esters to liver and steriodogenic tissues for metabolism and excretion. These functions of HDL, mediated by its major protein component, apolipoprotein A-I (apoA-I), underlie the antiatherogenic role of this lipoprotein class. In several key steps of HDL metabolism, apoA-I undergoes conformational changes to adapt to changing lipid contents of the HDL, to allow the binding of apoA-II to HDL, and possibly to mediate interaction with membranes and with lecithin cholesterol acyltransferase (LCAT). A putative "hinge" region has been implicated in the conformational changes of apoA-I. The specific aims of this project are to identify the helix(es) of apoA-I that are responsible for the "hinge" functions and the key amino acids that modulate the conformational adaptability of the "hinge" domain, and to study the structural and dynamic differences between the "closed" and "open hinge" forms of apoA-I. To identify the helix(es) involved in the "hinge" functions of apoA-I, we propose to construct and express in E. coli mutants of apoA-I with each of the candidate helixes replaced with the first or last helixes of apoA-I, which bind tightly to lipid and are not mobile. After structural studies of the mutants by spectroscopic (CD and fluorescence) methods and investigation of their lipid-binding properties, the apoA-I mutants in defined RHDL particles will be examined for their "hinge" functions: particle rearrangement, apoA-II binding, interaction with bilayer membranes, and LCAT activation. The identity of the "hinge" helix(es) will be confirmed and the structural rearrangements and their dynamics will be studied by constructing Cys mutants of apoA-I as sites for specific crosslinking or fluorescent labeling. The hydrophobicity, charge distribution, and role of Pro residues in the "hinge" helix(es) will be assessed by mutagenic substitution of individual amino acids. These studies are expected to localize the "hinge" region of apoA-I and to clarify its mechanism in several important functions of apoA-I.