MOLECULAR BASIS FOR APOLIPOPROTEIN AI FUNCTION

载脂蛋白A-I（apo A-I）是高密度脂蛋白（HDL）中发现的主要蛋白质，与HDL的所有已知生理功能有关，包括HDL对冠状动脉疾病和动脉粥样硬化的保护作用。该领域的一个重要问题是apo A-I结构在蛋白质从无脂质构象转化为脂质结合构象中所起的作用。这个问题一直是通过各种生物化学，生物物理和遗传技术进行大量测试的主题。用于解决这一问题的不同方法导致了几种竞争模型的开发，在其无脂质和脂质结合构象的载脂蛋白A-I的结构。对这些竞争模型的预测进行严格测试是此修订应用程序的重点。研究小组确定了载脂蛋白A-I突变体的第一个原子分辨率[Borhani，et al 1997]。该结构中包含的信息（目前正在3埃处进行细化）提供了一个框架，用于测试载脂蛋白A-I结构的各种竞争模型的预测。两个具体目标是：1。无脂质载脂蛋白A-I的生理相关结构是什么？(a)它是一个球状体，一个棒状体，还是两者都是？(b)它是否有一个离散的三级结构，或者它是一个没有明显三级结构的“熔融球”？2.脂质结合载脂蛋白A-I的构象是什么？(a)当与盘状脂质复合物结合时，它是一种离散的结构（描述为具有确定的螺旋间配对的“栅栏”或“带”），还是存在不太确定的构象，没有明显的三级结构或螺旋间配对？将采用多种方法来测试各种模型的预测，例如半胱氨酸交联、芘激基缔合物形成、荧光共振能量转移（FRET）、分析超离心、热力学稳定性、偏振衰减内反射傅里叶变换红外光谱（PATIR-FITR）和脂质结合动力学。从长远来看，这些研究将有助于确定哪些竞争模型可能与生理学相关，并帮助我们了解脂质和胆固醇代谢。

DESCRIPTION (Adapted from abstract): Apolipoprotein A-I (apo A-I) is the major protein found in high density lipoprotein (HDL) an has been implicated in all of the known physiological functions of HDL, including the protective effect of HDL against coronary artery disease and atherosclerosis. One important question in this field is the role that apo A-I structure plays in the conversion of the protein from its lipid-free to lipid-bound conformation(s). This question has been the subject of substantial testing by a variety of biochemical, biophysical, and genetic techniques. The diverse approaches used to address this question have led to the development of several competing models for the structure of apo A-I in its lipid-free and lipid-bound conformations. Rigorous testing of the predictions of these competing models is the focus of this revised application. The research team determined the first atomic resolution for an apo A-I mutant [Borhani, et al 1997]. The information contained in this structure (currently being refined at 3 angstroms) provides a framework for testing the predictions of the various competing models for apo A-I structure. The two specific aims are: 1. What is the physiologically relevant structure of lipid free apo A-I? (a) Is it a globule, a rod or both? (b) Does it have a discrete tertiary structure or is it a "molten globule" with no distinct tertiary structure? 2. What is the conformation of lipid-bound apo A-I? (a) Is it a discrete structure when bound to discoidal lipid complexes (described as either a "picket fence" or a "belt" with defined interhelical pairing), or are there less defined conformations that have no distinct tertiary structure or interhelical pairing? A variety of methods will be employed to test predictions of the various models, such as cysteine crosslinking, pyrene excimer formation, fluorescence resonance energy transfer (FRET), analytical ultracentrifugation, thermodynamic stability, polarized attenuated internal reflection Fourier transform infrared spectroscopy (PATIR-FITR), and lipid binding kinetics. In the long term, these studies will help determine which of the competing models may be physiologically relevant and help us understand lipid and cholesterol metabolism.