Jose L. Soulages - US grants

protein structure function; apolipoproteins; lipid metabolism; conformation; chemical binding; protein purification; site directed mutagenesis; Orthoptera; nuclear magnetic resonance spectroscopy; mass spectrometry; fluorescent dye /probe; high performance liquid chromatography; aminoacid analyzer; nucleic acid sequence;

DESCRIPTION (provided by applicant): Exchangeable apolipoproteins are essential structural and functional components of lipoproteins. The stability of the lipoprotein particles, the activity of lipolytic enzymes and lipid transfer proteins, and the binding of the lipoprotein to receptors are among the processes regulated by the properties of apolipoproteins, which bind to the lipoprotein lipid surfaces as well as to cell membranes. Apolipoproteins modulate all those 3rocesses by means of their lipid binding activity, rate and extent of binding, and their structure in the lipid-bound state. Therefore, the molecular properties of the exchangeable apolipoproteins define the physiological role of lipoproteins and the homeostasis of the lipid metabolism, in general. The understanding of the physiological role of a given apoLp depends on the knowledge of the structure and properties of the protein molecule in both the soluble and lipid-bound state(s). The long-term goals of this project are the lentification and characterization of the functional domains of apolipoproteins in the lipid-bound states. This proposal involves studies on the relationship between structure and function of two apolipoproteins: insect ,Apolipophorin-Ill and human apolipoprotein A-I. The specific aims of this proposal include: 1) Study of the organization of apoA-I molecules in discoidal HDL iipoproteins; 2) The study of the regions of human apoA-I that interact with lipid in high-density lipoproteins; 3) The study of the correlation between the structure of apoA-I in HDL particles and the function of the lipoproteins. 4) The study of the mechanism of association of apolipophorin-III with phospholipid monolayers and bilayers, including native lipoproteins and reconstituted model lipoproteins; Accomplishment of these objectives is necessary to fully understand the physiology and pathology of the metabolic pathways where apolipoproteins are involved. This includes understanding of the role of apolipoproteins in the metabolism of lipids and related pathologies, such as atherosclerosis, but it could also involve other kind of pathologies, such as Alzheimer's disease.

This award is for the acquisition of an ion trap mass spectrometer (MS) to support a diverse portfolio of basic research activities. These activities are united by their strong need for the MS/MS fragmentation capabilities, the superior sensitivity, and the ability to resolve and analyze peptide mixes using upstream in-line liquid chromatography. The MS will provide crucial insights into the structure, function, and regulation of five fundamentally important biomolecules, including the Midline-1 phosphoprotein, the lipid-droplet-associated protein-1, and apolipoprotein A-I. The MS will provide molecular insights into cellular differentiation and lipid transport processes. The MS will illuminate limitations to photosynthetic productivity, and it will contribute to the use of pectins in foodstuffs and as bio-industrial precursors. The MS will also be used to identify proteins governing plant-insect and plant-microbe relationships, protein chaperone networks, plant responses to oxidative stress, and plant meiosis. Because the most advanced MS on campus has no fragmentation, Liquid Chromatography, or Electro-Spray Ionization capabilities, the new MS will be valuable for many of the university's current research activities.

In addition to the scientific benefit, the MS will be useful for the investigators' undergraduates, graduate students, and postdoctoral trainees. All of these students and trainees will analyze data from the MS, and about a third of them will be trained to use this sophisticated instrument directly. The MS will enhance the existing semi-annual workshops on ""Mass Spectrometry and Proteomics,? and it will be integrated into the university's undergraduate biochemistry laboratory curricula to expose students to modern analytical approaches. The MS will be featured in outreach efforts that will include presentations on-campus and off-campus, as well as other novel outreach efforts. Approximately half of this outreach will be directed toward: minority programs, the minority institute Langston University, two of the State's non-Ph.D. institutes, and students who do not currently consider themselves research-bound.