In ischemia intracellular levels of fatty acids and their immediate metabolites, acylcarnitine and acyl-CoA, rise to abnormal levels. Normal transport and enzymatic processes appear to be insufficient to handle these high loads, and the consequent increased partitioning of these lipids into membranes can disturb membrane-related cell functions, such as contraction, excitability, rhythm, and cell viability. The same lipids are implicated in the pathology of genetic-linked disorders of fatty acid oxidation, which are important causes of cardiomyopathy and other debilitating diseases. The overall aim of this project is to describe structural and dynamic aspects of fatty acid binding and transport in plasma, in cell membranes, and in the cytosol. The overall strategy is to study native fatty acids with state-of-the-art methods in both structural and cell biology. The four specific aims are, first, to characterize interactions of fatty acids and their immediate metabolites (acyl-CoA and acylcarnitine) with model membranes and biological membranes by 13/C NMR spectroscopy and by titration calorimetry. The second specific aim is to elucidate molecular details of fatty acid interactions with albumin, primarily by X-ray crystallographic and multidimensional NMR studies of large fragments of albumin. The third specific aim is to elucidate the molecular details of intracellular fatty acid and lipid binding proteins by determining their solution NMR structure with and without ligands. The final specific aim is to study movement of fatty acids across membranes and desorption from membranes by fluorescence methods monitoring pH changes in cells and in vesicles. Our hypothesis of fatty acid-induced pH changes in cells will be examined by whole cell fluorescence and video imaging fluorescence measurements in single cells. This work will define certain important molecular interactions of fatty acids in the plasma compartment, in membranes and in the cytosol. It will permit a detailed comparison of the binding modes of albumin and intracellular fatty acid binding proteins and a better understanding of the functions of intracellular proteins. It will establish mechanisms of fatty acid permeation through membranes and provide a real time assay of fatty acid movement into cells. As models of physiologically important structures and processes, these in vitro systems will help form a basis for clarifying complex pathophysiological responses in ischemia. This work will also help understand the molecular basis of disorders of fatty acid metabolism, diabetes, and obesity.
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