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Thermodynamic Investigations of Metalloproteins: Metal as Probe and Protein as Probe
- Abstract:
Several metalloproteins, both metal transport proteins and the classic metalloprotein hemoglobin, were investigated using a variety of biophysical and electrochemical techniques. Thermodynamic measurements in each case provide insight into the role and mode of action of each metalloprotein under investigation.
Focusing on the thermodynamic properties of the heme-bound iron, we have investigated the structure-function relationships underlying two important physiological responses of hemoglobin: the Root Effect of hemoglobin from certain fish and the differential nitrite reactivities of hemoglobins from clams. The structural basis of these two phenomena is debated. We have measured the reduction potentials of these Hbs using spectroelectrochemistry (SEC) and compare our findings with oxygen binding studies performed by our collaborators. In both cases, our data strongly suggests that steric hindrance is the determining factor governing respective physiological responses of the hemoglobins.
Again using the metal as the probe, we have investigated the reduction potential of titanium bound by Transferrin (Tf). Tf is the human iron transport protein that can also bind titanium. To address the possible mechanisms of titanium transport through the hypothesized redox-mediated Fe2-Tf transport pathway, a modified SEC method was developed to measure the electrochemical properties of metalloproteins with very negative potentials. However, the reduction potential of Ti2-Tf is far too negative to access with our system. As an alternative, the redox properties of several model compounds were characterized in order to build a context to interpret the Ti2Tf results.
Shifting our focus to the thermodynamic properties of the protein, we have elucidated protein-protein and protein-metal interactions important for pathogenic bacteria. N. gonorrhoeae steal iron from their human host by expressing a receptor (TbpA/TbpB), which binds the human iron transport protein transferrin (Tf). Once iron crosses the outer membrane, Ferric Binding Protein (FbpA) transports it across the periplasm to the cytosol. We report the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA and demonstrate that both TbpA and TbpB individually can deferrate transferrin without energy supplied from TonB resulting in sequestration by apo-FbpA. Further, we have investigated the role of specific domains of TbpA in this iron uptake pathway and the specific effects of TbpA/TbpB on iron release from Tf in the absence of FbpA.
Ph.D. Dissertation Defense Examination Seminar
Student Exams Seminar