MOLECULAR BASIS FOR APOLIPOPROTEIN AI FUNCTION

载脂蛋白A-I(apo A-I)是高密度脂蛋白中的主要蛋白质，参与了高密度脂蛋白所有已知的生理功能，包括对冠状动脉疾病和动脉粥样硬化的保护作用。这个领域的一个重要问题是载脂蛋白A-I结构在蛋白质从无脂构象到脂结合构象的转换中所起的作用(S)。这个问题已经通过各种生化、生物物理和遗传技术进行了大量的测试。用来解决这个问题的不同方法导致了关于载脂蛋白A-I在其无脂构象和脂结合构象中的结构的几个相互竞争的模型的发展。对这些相互竞争的模型的预测进行严格测试是这一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，等人，1997]。该结构中包含的信息(目前以3埃精炼)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。两个特定的目的是：1.无脂载脂蛋白A-I的生理相关结构是什么？(A)它是球状、杆状还是两者兼有？(B)它是具有离散的三级结构，还是没有明显三级结构的“熔化球状”？2.脂类结合的载脂蛋白A-I的构象是什么？(A)当它与盘状脂质复合体结合时，它是一个离散的结构(被描述为“尖桩栅栏”或“带”，具有明确的螺旋间配对)，或者是否存在定义较少的构象，没有明显的三级结构或螺旋间配对？将使用各种方法来测试各种模型的预测，例如半胱氨酸交联法、芘准分子的形成、荧光共振能量转移(FRET)、分析性超速离心法、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相互竞争的模型可能是生理相关的，并帮助我们了解脂肪和胆固醇代谢。

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.