We propose to express recombinant full length and N- and C-terminal half- molecules of human serum transferrin (hTF) and hen ovotransferrin (oTF) and site-directed mutants and chimeras thereof in baby hamster kidney cells in sufficient quantities for studies of cellular function and chemico-physical characterization. Functional studies will address a) the ability of these proteins to bind to various metal ions and synergistic anions, b) the relative affinities and capacities of these proteins for cellular transferrin receptors and iron delivery to cells, e.g., chick embryo red cells, HeLa cells and c) the ability of these proteins to support division and growth of tissue cultured cells. Methods will include a) uv-vis, NMR and EPR spectroscopies, b) radioisotope labeling and c) cell culture. Physical studies will be used to probe structural changes in site- directed mutants and to correlate them with changes in function. EPR and uv-vis spectroscopies will allow assessment of changes in binding affinities for metal ions and synergistic anions and in the response of the proteins to """"""""inert salt"""""""". The latter effect bears on the mechanism of opening of the binding site cleft prior to release of metal ion. Multi-nuclear NMR studies will be broadened to include more site-directed mutant forms. Key to these experiments will be the biological incorporation of NMR silent, e.g., deuterated, and NMR sensitive, e.g., 19F-, 13C-, 15N-containing, amino acids. These studies will be used to monitor binding of synergistic and """"""""inert"""""""" anions, metal ions, hydrogen ions (pH titrations) and conformational changes in the proteins consequent to these additions. Site-directed mutagenesis will allow assignment of selected NMR resonances. Microcalorimetric experiments will allow assessment of thermodynamic properties and changes in these proteins. Large conformational changes will also be addressed by low- angle X-ray scattering.
Showing the most recent 10 out of 84 publications
