Professor, Department of Physiology & Biophysics
The goal of our research is to determine protein function through the investigation of intermediate state structures generated along a given reaction pathway. These intermediate states involve transient forms of the protein, cofactor and substrate. In a variety of enzyme systems, intermediates include paramagnetic species in the form of metals and/or organic radicals. In addition, for systems which lack endogenous paramagnetic species, it is often advantageous to introduce a stable radical "spin label" to serve as a reporter of protein structure and dynamics. Electron paramagnetic resonance (EPR) spectroscopy is well suited for the characterization of all of these classes of paramagnetic species. Thus our primary experimental tools for structural characterization involve advanced EPR techniques, including electron spin echo envelope modulation (ESEEM), electron nuclear double resonance (ENDOR), and Hyperfine Correlation Spectroscopy (HYSCORE). Pulsed electron double resonance (PELDOR) techniques are used to measure distances between mutagenically introduced spin labels out to 50 Å. Quantum mechanical simulations of experimental spectra are developed from first principles for the accurate determination of spectral parameters. For proteins which are not amenable to NMR or crystallographic techniques, homology modeling and molecular dynamics calculations are used to generate structures which can be tested using EPR techniques. Instrumentation and spectroscopic techniques are implemented as required by the systems under investigation. A primary focus in this regard is the development and application of high frequency cw and pulsed EPR/ENDOR spectroscopy. HF-EPR/ENDOR extends EPR spectroscopy to high magnetic field strengths and enhances the capabilities of the technique to determine molecular and electronic structure.
Examples of projects currently under study include: structure of protein active sites and substrate intermediates in coenzyme B12-dependent ribonucleoside triphosphate reductase; identity and structure of radicals generated during catalysis by prostaglandin H synthase (COX or PGHS) and cytochrome c oxidase; molecular structure determination of kinesins using spin-labeling techniques; HF-EPR/ENDOR of a variety of protein and substrate-derived radical species.
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Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Avenue
Ullmann Building, Room 225
Bronx, NY 10461