APPLICATION OF MASS SPECTROMETRY TO STRUCTURAL BIOLOGY

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Recent instrumental developments in mass spectrometry, such as matrix- assisted laser desorption and electrospray ionization, have enabled mass spectrometrists to investigate biological molecules with significantly higher Mr than previously possible. The combination of these techniques with chemical processing of large biopolymers such as proteins now enables mass spectrometry to play a significant role in the realm of structural biology. We are currently working on several projects: modifications on apolipoprotein A-1 (detailed below); developing the capability of probing non-covalent interactions between proteins and between proteins and DNA using mass spectrometry; and determining the cysteine residue in the insulin receptor that is susceptible to biotinylation. One project we are currently working on is the identification of the post-translational modifications, especially sites of fatty acid acylation, on apolipoprotein A-I secreted by Hep G2 human hepatoma cells. Apolipoprotein A-I (apo A-I) is the principal protein present in human high density lipoproteins. It not only serves as a structural protein for the integrity of the lipoprotein particle, it also is a cofactor for the enzyme lecithin cholesterol acyltransferase (LCAT) that esterifies cholesterol in the blood. Apo A-I has been proposed to play an important role not only in transporting cholesterol from nonhepatic tissues to the liver, but it may also be important in preventing the development of atherosclerosis. The 9 amphipathic alpha helices present in antiparallel array in this protein are critical for lipid binding, coactivator function of LCAT, and potentially for interaction with the cellular surface in order to facilitate cellular cholesterol transport. Because nascent, but not circulating, apo A-I is fatty acid acylated, Hoeg has proposed that the esterification of specific residues with fatty acids may be critical for protecting the hepatocyte and enterocyte, which secrete apo A-I into the circulatory system. Apo A-I is acylated only in primary hepatocyte cultures and in well-differentiated hepatoma cell lines indicating that acylation requires specific cellular factors. Transfected nonhepatic cell lines synthesize and secrete apo A-I without fatty acid acylation as defined by radio labeled fatty acid studies. These data indicate potential role(s) for the addition of these prosthetic groups. This could be altering the ability for apo A-I to induce transmembrane signaling and/or by altering the removal of cholesterol from intracellular membrane pools. Finally, nascent apo A-I may require fatty acyl groups for initial HDL particle assembly. By identifying the specific amino acids acylated, site-directed mutagenesis of apo A-I can be used to determine the physiological significance of acylation.