Explorations of the relationship between macromolecular structure and function are a key element of modern biochemistry. Our group uses a combination of X-ray crystallography and site-directed mutagenesis to examine this relationship. Determination of a target enzyme's structure provides the starting point. This is followed by iterative cycles of site-directed mutagenesis and characterization of the effects of the induced mutation on enzyme structure and function. In this way it is possible to identify residues that are critical for catalysis; to evaluate the energetics of enzyme:substrate interactions; to examine the effect of the local environment on reactivity: and to engineer new or modified reactivities into the parent enzyme.
Enzymes currently under study in our laboratory include cytochrome c peroxidase, DNA polymerase beta, and dihydrofolate reductase. A selective bibliography follows. You can also choose to browse some abstracts from selected recent work.
"Crystal Structure of a Complex Between Electron Transfer Partners, Cytochrome c Peroxidase and Cytochrome c" Pelletier, H., & Kraut, J. Science 258, 1748-1755 (1992).
"Histidine 52 is a Critical Residue for Rapid Formation of Cytochrome c Peroxidase Compound I" Erman, J.E., Vitello, L.B., Miller, M.A., Shaw, A., Brown, K.A., & Kraut, J. Biochemistry 32, 9798-9806 (1993).
"Reaction of Horse Cytochrome c with the Radical and the Oxyferryl Heme in Cytochrome c Peroxidase Compound I" Hahm, S., Miller, M. A., Geren, L., Kraut, J., Durham, B., & Millett, F. Biochemistry 33, 1473-1480 (1994).
"2.2 Angstrom Structure of Oxy-peroxidase as a Model for the Transient Enzyme:Peroxide Complex" Miller, M.A., Shaw, A., & Kraut, J. Nature Structural Biology 1, 524-533 (1994).
"Interaction Domain for the Reaction of Cytochrome c with the Radical and the Oxyferryl Heme in Cytochrome c Peroxidase Compound I" Miller, M.A., Liu, R.-Q., Hahm, S., Geren, L., Hibdon, S., Kraut, J., Durham, B., & Millett, F. Biochemistry 33, 8686-8693 (1994).
"A Cation Binding Motif Stabilizes the Compound I Radical of Cytrochrome c Peroxidase" Miller, M.A., Han, G.W. & Kraut, J. Proc. Natl. Acad. Sci. USA 91, 11118-11122 (1994).
"Regulation of Interprotein Electron Transfer By Trp 191 of Cytochrome c Peroxidase" Miller, M.A., Vitello, L. B., & Erman, J. E. Biochemistry 34, 12048-12058 (1995).
"Identifying the Physiological Electron Transfer Site of Cytochrome c Peroxidase by Structure-Based Engineering" Miller, M. A., Geren, L., Han, G. W., Saunders, A., Beasley, J., Pielak, G., Durham, B., Millett, F., & Kraut, J. Biochemistry 35, 667-673 (1996).
"Crystal Structure of Rat DNA Polymerase Beta: Evidence for a Common Polymerase Mechanism" Sawaya, M.R., Pelletier, H., Kumar, A., Wilson, S. H., & Kraut, J. Science 264, 1930-1935 (1994).
"Structures of Ternary Complexes of Rat DNA Polymerase Beta, a DNA Template-Primer, and ddCTP" Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. Science 264, 1891-1903 (1994).
Crystal Structures of Human DNA Polymerase Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity Huguette Pelletier, Michael R. Sawaya, William Wolfle, Samuel H. Wilson, and Joseph Kraut (1996a) Biochemistry, Volume 35, Issue 39, pp. 12742- 12761.
A Structural Basis for Metal Ion Mutagenicity and Nucleotide Selectivity in Human DNA Polymerase Huguette Pelletier, Michael R. Sawaya, William Wolfle, Samuel H. Wilson, and Joseph Kraut (1996b) Biochemistry, Volume 35, Issue 39, p 12762-1277.
Characterization of the Metal Ion Binding Helix-Hairpin-Helix Motifs in Human DNA Polymerase by X-ray Structural Analysis Huguette Pelletier and Michael R. Sawaya (1996c) Biochemistry, Volume 35, Issue 39, p 12778-12787.
"Determination by Raman Spectroscopy of the pKa of N5 of Dihydrofolate Bound to Dihydrofolate Reductase: Mechanistic Implications" Chen, Y.-Q., Kraut, J., Blakely, R. L., & Callender, R. Biochemistry 33, 7021-7026 (1994).
"Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications" Reyes, V. M., Sawaya, M., Brown, K. A., & Kraut, J. Biochemistry 34, 92710-2723 (1995).
"Crystal Structures of Escherichia coli Dihydrofolate Reductase Complexed with 5-Formyltetrahydrofolate (Folinic Acid) In Two Space Groups - Evidence for Enolization of Pteridine O4" Lee, H., Reyes, V. M., & Kraut, J. Biochemistry 35, 7012-7020 (1996).
Loop and Domain Movements in the Mechanism of E. Coli Dihydrofolate Reductase: Crystallographic Evidence. M. R. Sawaya, & J. Kraut. Biochemistry, Vol 36, pp. 586-603, (1997).