Research in our laboratory is focused on interactions between DNA, RNA and proteins. Our goal is to understand the step-wise structural and energetic changes associated with these binding events.
Dye molecules attached to oligonucleotides and proteins act as reporter groups, sensitively signaling structural changes that occur on the nanosecond time scale. A particularly powerful fluorescence technique is fluorescence resonance energy transfer (FRET), in which there is a non-radiative transfer of energy between a donor dye molecule and an appropriate acceptor molecule. The efficiency of energy transfer is highly dependent upon the distance between the donor and acceptor, allowing determination of distances and distance changes from 40 to 80 Å with precision well within 1 Å. A14-mer oligonucleotide has a fluorescein on the 3' end that functions as a FRET donor and a rhodamine derivative on the 5' end that functions as an acceptor in energy transfer. We use such molecules to monitor conformational changes induced in DNA upon protein binding. We have recently developed a new technique acceptor detected FRET (ADFRET) which allows us to measure distances between dyes out to 100 Å from time-resolved fluorescence spectra. This technique should allow direct comparisons of distances by FRET and various scanning microscopies.
Lawrence J. Parkhurst, “Distance Parameters Derived from Time Resolved FRET Measurements and Their Use in Structural Interpretations of Thermodynamic Quantities Associated with Protein-DNA Interactions”, Methods in Enzymology, G. Ackers, ed. 372, 235-262 (2004).
I. B. Matheson, L. J. Parkhurst, and R. J. DeSa, “Efficient Integration of Kinetic Differential Equation Sets Using Matrix Exponentiation”, Methods in Enzymology, G. Ackers ed., 384, 18-39 (2004).
K. M. Masters, K. M. Parkhurst, M. A. Daugherty, L. J. Parkhurst, “Native Human TATA Binding Protein Simultaneously Binds and Bends Promoter DNA Without a Slow Isomerization Step or TFIIB Requirement”, J. Biol. Chem. 278, 31685-31690 (2003).
Lawrence J. Parkhurst, Kay M. Parkhurst, Robyn Powell, Jiong Wu, and Sarah Williams. “Time-Resolved Fluorescence Resonance Energy Transfer Studies of DNA Bending in Double-Stranded Oligonucleotides and in DNA-Protein Complexes”, Biopolymers (Nucleic Acid Sciences) 61, 180-200.
Philip R. Hardwidge, Jiong Wu, Sarah L. Williams, Kay M. Parkhurst, Lawrence J. Parkhurst, and L. James Maher, III. “DNA Bending by bZIP Charge Variants: A Unified Study Using Electrophoretic Phasing and Fluorescence Resonance Energy Transfer”, Biochemistry 41, 7732-7742 (2002).