Department of Chemistry

Mary D. Barkley

Mary D. Barkley

Chair of the Department of Chemistry
M. Roger Clapp University Professor of Arts & Sciences

216.368.0602      mdb4@case.edu      Clapp 202

Interests: Analytical Chemistry, Biochemistry, Biophysical Chemistry, Medicinal Chemistry, Photochemistry, Physical Chemistry, Theoretical Chemistry

B.S., Stanford University, 1964
Ph.D., University of California, San Diego, 1969
NIH Postdoctoral Fellow,
Salk Institute for Biological Studies, 1969-1972

Our research group investigates the structure and dynamics of biological macromolecules. With the near completion of the human genome project, attention is turning to the proteins that are encoded by the genome. An important feature of proteins is their structure. Specifically, it is important to understand how protein structure is related to protein function. A better understanding of protein structure and function makes it possible to design better drugs to combat disease. Our laboratory is using fluorescence spectroscopy to understand how protein structure affects protein function.

After a molecule absorbs a photon of light, a few billionths of a second later it emits a photon of visible light called fluorescence. We use that fluorescent light as a probe to follow changes in protein structure that are important for protein function. A number of biological molecules are naturally fluorescent. In addition, we can attach fluorescent probes to engineered proteins and synthetic peptides or oligonucleotides.

Macromolecular recognition is the basis of biological specificity. We are investigating conformational changes in proteins and DNA important in recognition. EcoRI endonuclease recognizes and cleaves its recognition sequence on DNA with high specificity. We are determining the structure and dynamics of the N-terminal region of the endonuclease, which is essential for cleavage activity but appears disordered in the crystal structure of the endonuclease-DNA complex. Site-directed mutagenesis is used to introduce fluorescent probes at specific positions in the N-terminal region of the protein. Changes in conformation and flexibility of the DNA recognition site are probed using the fluorescent base 2-aminopurine. Photophysical studies of 2-aminopurine riboside and 2-aminopurine-substituted oligonucleotides provide a rigorous framework for interpreting fluorescence changes in structural terms.

Viral replication is a key target for drug therapy. We are studying enzymes from two viruses: the AIDS virus HIV-1 and hepatitis C virus HCV. The first step in replication of the HIV genome is to copy the single-stranded viral RNA into double-stranded DNA. The enzyme that does this is reverse transcriptase, a heterodimer with two activities: DNA polymerase and RNA cleavage.

We are studying the conformational changes in reverse transcriptase on RNA and DNA substrates using pre-steady state kinetics and fluorescence techniques. Heterodimer formation is examined by analytical ultracentrifugation. These experiments involve site-specific fluorescent labeling of reverse transcript as well as RNA and DNA substrates containing fluorescent pteridine base analogs.

The first step in replication of the HCV genome is to copy the single-stranded viral RNA into double-stranded RNA. NS5B RNA polymerase synthesizes the viral RNA, but may require other viral proteins and also host cell proteins. We are studying the initiation of synthesis of the second RNA strand by NS5B polymerase and the effect of other viral enzymes on this process by molecular biological and biophysical techniques, including footprinting, fluorescence spectroscopy, and analytical ultracentrifugation.

Tryptophan is a fluorescent amino acid. It is widely used to monitor protein conformation, because its fluorescence is so sensitive to the local enviromnment of the indole chromophore. However, the relationship between tryptophan fluorescence and protein structure is poorly understood. We are developing structural interpretations for the complex fluorescence decays of tryptophan using stereochemically constrained peptides and peptides bound to proteins. The effects of other protein functional groups on tryptophan fluorescence is examined in small rigid peptides. The structure of the peptides is determined by X-ray crystallography, molecular mechanics, and multidimensional NMR.

Selected Publications
  • "Intramolecular Quenching of Tryptophan Fluorescence by the Peptide Bond in Cyclic Hexapeptides. " Paul D. Adams, Yu Chen, Kan Ma, Michael G. Zagorski, Frank D. Sönnichsen, Mark L. McLaughlin, and Mary D. Barkley, J. Am. Chem. Soc., 124, 9278-9286 (2002).
  • "RNA Polymerase Alters the Mobility of an A-Residue Crucial to Polymerase-Induced Melting of Promoter DNA." Biochemistry, 41, 15334-15341 (2002).