2003 Beckman Scholars at Carnegie Mellon
|Mary Ellen Wiltrout, Department of Biological Sciences, Carnegie Mellon University
(Advisor: Dr. Chien Ho)
A Biophysical Investigation of Recombinant Hemoglobins with Mutations in the Distal Heme Pocket
To investigate the effect on the structural and functional properties of hemoglobin, amino acid substitutions have been made in the distal side of the heme pocket for both the a- and b-chains of human normal adult hemoglobin (Hb A). Using an Escherichia coli expression system, we have constructed and expressed three recombinant hemoglobins, rHb (αL29W), rHb (βL28W), and rHb (βL28F). The α29 and β28 residues are located in the B10 helix of the α- and β-chains of Hb A, respectively. All three recombinant hemoglobins exhibit very low oxygen affinity and reduced cooperativity as compared to those of Hb A. Low oxygen affinity and high cooperativity are important properties for possible Hb-based blood substitutes, but in general, low oxygen affinity in natural mutant Hbs is accompanied by an increased rate of autoxidation. Hemoglobin is biologically functional in the reduced state. The distal heme pocket of the B10 helix is a location of interest for genetic modification, since in previous studies an α-chain B10 mutation, α29F, in myoglobin and hemoglobin showed autoxidation inhibition and the ability to lower NO-induced oxidation. Because of these oxidation properties found in rHb (αL29F), a high oxygen affinity mutant in the B10 helix, the autoxidation and azide-induced oxidation rates were determined for these three low oxygen affinity rHbs in the B10 helix. Autoxidation and azide-induced oxidation were monitored by visible spectrophotometry in the 400 nm to 700 nm range to determine the percentage of ferrous-Hb as a function of time. All three rHbs had increased rates of autoxidation and azide-induced oxidation compared to Hb A, but the rates for rHb (αL29W) were about half of either rHb with a mutation in the β28 position. 1H-NMR showed the α1β1 and α1β2 subunit interfaces in both deoxy and liganded states are not perturbed in these three rHbs. The tertiary structures around the heme pockets of the mutated chains are perturbed in these three rHbs as expected. The reaction of hemoglobin with nitric oxide and the subsequent depletion of nitric oxide have been shown to be the cause of hypertension in protein-based blood substitutes. Therefore, NO-induced oxidation rates and NO reaction rates will be measured on the stopped-flow apparatus to further characterize these three rHbs.
|Margaret Young, Department of Biological Sciences, Carnegie Mellon University
(Advisor: Dr. Jonathan Minden)
Proteomic Analysis of Cell Shape Changes During Gastrulation in Drosophila
My research project involved the study of proteins and mechanisms involved in cell shape changes during development. A Drosophila model was used to study the embryonic process of ventral furrow formation. This process involves a series of cell shape changes that cause the formation of the mesoderm layer of the embryo. Mutational analysis has identified several genes that are involved in a signaling cascade that triggers ventral furrow formation, but this approach has not identified any cytoskeletal proteins or structural proteins involved in this process. An important thing to remember is that the embryo contains maternally contributed proteins, in addition to zygotically expressed genes, and that many proteins are regulated by post-translational modification. For this reason, a direct protein analysis may be more useful than genetic screens for identification of the protein changes that cause the furrow to form. For this purpose, our lab has developed a new approach to identify protein changes between different cells. The technique, difference gel electrophoresis (DIGE), is a modified two-dimensional polyacrylamide gel electrophoresis method. Two protein samples are labeled separately with different fluorescent dyes. The samples are then mixed and analyzed. The mixed sample is first run on an isolelectric focusing gel to separate proteins on the basis of charge; the second dimension is an SDS-PAGE to separate the proteins on the basis of size. The gel is then fluorescently imaged to determine whether there are any protein differences. If a protein is the same in both cell samples, there will be one spot with both colors. If a protein is found in only one sample, then the spot will show only one of the dyes. This method can identify differences in level of expression as well as post-translational changes, such as phosphorylation or proteolysis, which might change the protein in such a way that makes ventral furrow formation possible. Such changes cannot be identified through RNA analysis. The spots that change are called ³difference-proteins². Once these proteins are detected with DIGE, they are identified by mass spectrometry (MS). Thus far, over fifty difference-proteins have been found, and many of them have been identified. My project was to determine the function of four of these proteins in ventral furrow formation and cell shape change. The proteins that I studied were determined to be time-dependent in ventral furrow formation by DIGE: belle, eIF-4e, squid and a dynamin-like protein. I used time-lapse microscopy to monitor the effect of reducing protein abundance with RNA interference. In the future I will also examine proteome changes in response to altered amounts of the proteins-of-interest. This will allow us to assemble a molecular framework for the proteins involved in driving cell shape change during ventral furrow formation.