MOLECULAR BASIS FOR APOLIPOPROTEIN AI FUNCTION

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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.