CAREER: Spectroscopic Studies of Functionally Significant Interactions in Iron-dependent Proteins and Enzymes

University Of Miami, Coral Gables FL

Investigators

Abstract

With this CAREER award, the Chemistry of Life Processes Program in the Chemistry Division is funding Katlyn Meier from the University of Miami to investigate protein-protein interactions in iron-containing metalloproteins and metalloenzymes using a metal-centric spectroscopy approach. Protein-protein interactions define how proteins bind to and interact with each other to affect their structures and how they function. Dr. Meier’s research will study the interactions involving two different proteins that contain iron by using methods that follow specifically the chemical properties of this metal at the protein-protein interaction surface. These spectroscopic methods, when coupled with computation, will provide information on interaction interfaces among proteins under conditions that are less artificial and more similar to those found in cells. One of these iron containing proteins interacts with other similar proteins to control the synthesis of important molecules, including vitamins and hormones, or inhibit metabolism of drugs. The second iron protein being studied assembles into a complex composed of four identical copies of itself. This tetramer protein is a catalyst that helps produce compounds that reduce effects of oxidants in cells. The project will provide training to graduate and undergraduate students in advanced biochemical, biophysical, and computational methods. Finally, the project includes an integrated outreach program aimed at introducing middle and high school students to the basics of spectroscopy that includes a mentorship program aimed at increasing retention of future scientists from underserved populations. Characterization of the protein-protein interaction (PPI) interface and the role of the metals requires an interdisciplinary approach that employs several spectroscopic techniques. Low temperature electron paramagnetic resonance (EPR) spectroscopy will be used to selectively interrogate the structural and electronic properties of the iron sites and to provide feedback on the paramagnetic species in each sample. Variable temperature, variable field Mössbauer spectroscopy will be used to conclusively determine iron or heme oxidation state and to measure coupling between iron centers. Changes in secondary and quaternary structures that occur as the result of protein conformational changes induced by the PPI will be monitored by far-UV circular dichroism (CD), while CD in the visible and near-infrared regions will report on changes in charge transfer bands and d-d centered transitions relevant to perturbations in the metal-protein complex. Finally, fluorescence measurements will provide additional feedback about the interaction surface via intrinsic tryptophan fluorescence quenching, among other methods. The combination of these techniques is expected to provide for greater understanding of global and local electronic and structural changes associated with PPIs by spanning a wide range of energy scales that, in turn, probe conformational changes at various distance scales. Insight gained from this work will be used to calibrate computational models of PPIs. It is anticipated that the proposed studies will provide spectroscopically informed insight that is currently lacking in the field, but yet is required for “bottom-up” design of peptidomimetics to modulate and report on PPIs. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.