About

Interests

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

Education

• BS, Stanford University, 1964
• PhD, University of California, San Diego, 1969
• NIH Postdoctoral Fellow, Salk Institute for Biological Studies, 1969-1972

About

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. EcoRIendonuclease 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 the AIDS virus HIV-1. 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.

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

“Allosteric Suppression of Structural Dynamics of HIV-1 Reverse Transcriptase upon Inhibitor Binding.” James M. Seckler, Mary D. Barkley, and Patrick L. Wintrode, Biophys. J., 100, 144–153 (2011).

“Correlation of Tryptophan Fluorescence Spectral Shifts and Lifetimes Arising Directly from Heterogeneous Environment.” Chia-Pin Pan, Pedro L. Muiño, Mary D. Barkley, and Patrik R. Callis, J. Phys. Chem. B, 115: 3245–3253 (2011).