Computational and Experimental Studies of Structure/ Dynamics of HDL Assemblies

该项目的目标是继续我们对HDL的计算和实验研究，重点是从PL-贫apoA-I组装富含磷脂（PL）和富含胆固醇酯（CE）的HDL的机制。这些目标是由我们的主要假设驱动的，即apoA-I是一种独特的弹性双层结合蛋白，能够以小分子增量吸收PL和CE。为了实现这些目标，我们提出，使用分子动力学（MD）和实验方法的组合，按优先顺序列出的两个具体目标：1）研究富含PL的HDL的结构/动力学。为了实现这一目标，我们将： 100：2和50：2颗粒的更稳健的模拟，ii）通过改变注册和经由MD模拟和实验性地经由Cys突变的二聚化抑制埋藏的盐桥来研究旋转异构体注册的作用，iii）通过模拟和实验研究确定全长apoA-I的柔性结构域的作用和未酯化的胆固醇（UC）和/或鞘磷脂（SM）的作用，脂质头基和apoA-I之间的盐桥形成的作用，以及肽模拟物与富含PL的HDL的相互作用的机制，iv）通过模拟使用cryoEM和低角X射线散射成像富含PL的HDL组件 2)探讨贫磷脂apoA-1组装成富磷脂HDL的分子基础。为了实现这一目标，我们将使用：用途：i）来自最近的X射线晶体结构的单体apoA-1和使用颗粒收缩方法的二聚体PLD贫乏HDL的MD模拟; ii）实验方法以：确定从PL-贫乏的HDL组装富含PL的HDL的化学计量和动力学，确定来自血浆的前β（PL-贫乏）HDL的组成/化学计量，并确定由ABCA 1途径产生的富含PL的HDL的结构/组成。初步结果表明，这些目标中的大多数可以在所要求的支助的5年内实现。一个长期的目标，没有资金的要求是：确定的结构/动态CE丰富的HDL。由于MD模拟提供HDL的超分子图像的非凡力量，如我们的初步结果所示，通过结合MD模拟和实验方法，我们处于独特的地位，以获得新的见解的各种HDL亚种的结构/功能。作为一个长期目标，从所产生的动态结构-功能图，药物将被设计成修改“好”胆固醇，HDL，以预防或逆转冠心病。

The goals of this project are to continue our computational and experimental investigations of HDL, focusing on mechanisms of assembly of phospholipid (PL)-rich and cholesteryl ester (CE)-rich HDL from PL-poor apoA-l. These goals are driven by our lead hypothesis that apoA-l is a uniquely elastic bilayer-bounding protein capable of absorbing PL and CE in small molecular increments. To achieve these goals, we propose, using a combination of molecular dynamics (MD) and experimental approaches, two specific aims listed in order of priority: 1) To study the structure/dynamics of PL-rich HDL. To achieve this aim, we will: i) Perform more robust simulations of the 100:2 and 50:2 particles, ii) Study the role of rotamer registry by varying registry and inhibiting buried salt bridges via MD simulations and experimentally via dimerization of Cys mutations, iii) Determine the role of the flexible domain of full length apoA-l and the effect of unesterified cholesterol (UC) and/or sphingomyelin (SM) via simulations and experimental studies, the role of salt bridge formation between lipid headgroups and apoA-l, and the mechanisms of interaction of peptide mimetics with PL-rich HDL via simulations, iv) Image PL-rich HDL assemblies using cryoEM and low angle x-ray scattering 2) To explore the molecular basis for the assembly of PL-rich HDL from PL-poor apoA-l. To achieve this aim, we will use: i) MD simulations of monomeric apoA-l from a recent x-ray crystal structure, and dimeric PLD-poor HDL using the particle shrinkage approach; ii) experimental approaches to: determine stoichiometry and kinetics of assembly of PL-rich from PL-poor HDL, determine composition/stoichiometry of pre-beta (PL-poor) HDL from plasma, and determine the structure/composition of PL-rich HDL produced by the ABCA1 pathway. Preliminary results suggest that the majority of these aims can be achieved within the 5 years of requested support. A long-term aim for which no funds are requested is: To determine the structure/dynamics of CE-rich HDL. Because of the extraordinary power of MD simulation to provide supramolecular images of HDL as indicated by our preliminary results, by combining MD simulations and experimental approaches, we are uniquely positioned to gain new insights into the structure/function of the various HDL subspecies. As a long term goal, from the resulting dynamic structure-function maps, drugs will be designed to modify the "good" cholesterol, HDL, to prevent or reverse coronary heart disease.