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Biological Sciences  John W. Shriver Professor, Chemistry and Biology & Director Bimolecular NMR Laboratory    |
Research area: Structural Biology and Biophysics Research description: Many
DNA-binding proteins distort the structure of DNA, often with
significant changes in the twist and bend angles of the duplex.
Distortions in the binding site and beyond are necessary for DNA
packaging, as well as the regulation and expression of many genes of
medical relevance. Although about 500 structures of protein-DNA
complexes have been determined, our understanding of the interplay
between the energetics of binding and distortion is very much in its
infancy. This limitation stems from a number of factors, not the least
of which is ambiguities derived from protein instability since accurate
descriptions of the energetics of binding require data collection over
a temperature range that is inaccessible with many complexes. In
addition, ideally it should be possible to not only measure distortion,
but also manipulate and control it so that the energetics of distortion
can be directly related to structural changes. We use a novel approach
to this problem by developing and characterizing two model systems that
are well-suited for measuring the energetics of DNA binding and
distortion. These are obtained from the hyperthermophile Sulfolobus and
are similar to proteins with direct biomedical relevance which have
proven to be difficult to characterize energetically. The first
protein, Sac7d, is a small chromodomain protein similar to the
DNA-binding domain of HIV-1 integrase. Sac7d binds to the minor groove
of DNA and induces one of the largest known kinks in DNA with
intercalation of two amino acid side chains. NMR, fluorescence
resonance energy transfer (FRET), and calorimetry (ITC and DSC) are
being used to quantitatively characterize the role of protein residues
and DNA sequence in defining the structure of the complex and the
energetics of distorting DNA. The second protein, Sso10a, is a dimer of
winged helices that is being used to test the generality of the
conclusions drawn from Sac7d. This is the the first calorimetric study
of DNA binding by a winged helix, a fold important in many human
transcription factors. This is a basic research project which will
provide a description of the energetics of DNA interactions and
distortions that are important in many disease-related protein-DNA
complexes. The results will enhance our ability to rationally control
protein-DNA binding interactions in pharmacology and therapeutics. 1. W.P. Peters, S.P.
Edmondson, and J.W. Shriver
(2005) "The Role of Intercalating Residues in the Energetics of
Sac7d-DNA Binding and Bending" Biochemistry (in press). 5. Stephen Edmondson
and John W. Shriver (2004)
"DNA-Binding Proteins Sac7d and Sso7d from Sulfolobus" Methods in
Enzymology 334, 129-145. 6. .P. Edmondson, M. Kahsai, R. Gupta, and J.W. Shriver. (2004) "Characterization of Sac10a, a Hyperthermophile DNA Binding Protein from Sulfolobus acidocaldarius" SBiochemistry 43, 13026-36. 7. L. Chen, L.-R. Chen, W. Zhou, Y. Wang, M. Kahsai, A.T. Clark, S.P. Edmondson, Z.J. Liu, J.P. Rose, B.-C. Wang, E.J. Meehan, J.W. Shriver (2004) "The Hyperthermophile Protein Sso10a is a Dimer of Winged Helix DNA-Binding Domains Linked by an Antiparallel Coiled Coil Rod" J. Mol. Biol. 341, 73-91. 8. A.T. Clark, B.S.
McCrary, S.P. Edmondson, and J.W.
Shriver (2004) "Thermodynamics of Core Hydrophobicity and
Packing in the Hyperthermophile Proteins Sac7d and Sso7d" Biochemistry
43, 2840-53. |
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