UM Biophysics Faculty | Carol Fierke

Faculty Home Biophysics Home UM Home

Carol Fierke

Biological Catalysis; Molecular Recognition, Directed Evolution

Carol Fierke

Professor, Biological Chemistry and Chemistry

Research Scientist, Biophysics

Ph.D., Brandeis University

Dept: Biological Chemistry and Chemistry

Office Address: 4525 Chemistry

Phone: (734) 936-2678

Email: fierke@umich.edu

Carol Fierke's faculty website

Our overall research goal is to understand the mechanisms used by biological catalysts, both proteins and nucleic acids, to achieve high efficiency and stringent specificity. To this end, we probe structure-function relationships in enzymes using a combination of molecular biology techniques, thermodynamic and kinetic methods, and spectroscopic methods.

We are currently elucidating catalytic mechanisms and essential active site features of several medically important metalloenzymes, including protein farnesyltransferase, UDP-3-O-acyl-GlcNAC deacetylase and histone deacetylase. Inhibitors of these enzymes may be useful for the treatment of cancer and bacterial infections. To understand the role of proteins in modulating the reactivity of bound zinc, we are elucidating detailed structure-function relationships using mutagenesis, kinetic analysis, X-ray crystallography, and NMR spectroscopy. In conjunction with spectral and structural studies, these experiments should enhance our understanding of catalytic zinc sites and our ability to design potent inhibitors for these enzymes. Additionally, we are investigating the biological importance of these posttranslational modifications (prenylation and acetylation). Finally, we are developing methods to identify novel metal sites in proteins to elucidate the yeast “metallome”.

Our understanding of biological catalysis can be tested by the rational design or redesign of an enzyme. To this end, we are redesigning the affinity and specificity of the metal binding site in human carbonic anhydrase II. These enzyme variants are useful for optimizing a CAII-based fluorescent biosensor to measure and image metal ions in complex mixtures, including plasma, seawater and cells. We are using these imaging techniques to probe zinc homeostasis and zinc signaling in vivo in yeast and E. coli. Additionally, we are using "directed evolution" approaches to obtain altered enzyme function. Currently we use in vitro evolution methods to prepare and identify aldolase variants with novel substrate specificities. Characterization of the structure and function of these novel proteins will provide insights into catalysis, molecular recognition and molecular evolution.

We also investigate the structure, mechanism and substrate specificity of ribonuclease P (RNase P), a ribonucleoprotein complex that catalyzes the cleavage of tRNA precursors, an essential step in tRNA maturation. We are currently elucidating the structure of the RNase P holoenzyme using crosslinking, site-specific cleavage, crystallography, fluorescence resonance energy transfer and NMR spectroscopy. Furthermore, we are probing the catalytic mechanism of hydrolysis, including the role of magnesium ions, using structure-function relationships and isotope effects. These studies are increasing our understanding of the catalytic modes used by ribozymes in comparison to protein catalysts.

AWARDS

Chair-elect, 2005-2006, Biological Chemistry Division, American Chemical Society
Program Chair, 2003, ACS Meeting, Biological Chemistry Division
Chair; Enzymes, Coenzymes and Metabolic Pathways Gordon Conference Editorial Board, RNA
American Heart Association Established Investigator Award
David and Lucile Packard Foundation Fellowship
American Cancer Society Junior Faculty Research Award
National Institutes of Health Postdoctoral Fellow
UM Distinguished Faculty Achievement Award - 2005
Sarah Power Goddard Award, Univ. Mich. Women’s Caucus - 2005

Representative Publications

1) McCall, K. A. and Fierke, C. A. (2004) Probing Determinants of the Metal Ion Selectivity in Carbonic Anhydrase using Mutagenesis, Biochemistry 43, 3979-3986.

2) Bowers, K. E. and Fierke, C. A. (2004) Positively charged side chains in protein farnesyltransferase enhance catalysis by stabilizing the formation of the diphosphate leaving group, Biochemistry 43, 5256-5265.

3) Hartman, H. L., Bowers, K. E. and Fierke, C. A. (2004) Lysine 311 of Protein Geranylgeranyltransferase Type I Partially Replaces Magnesium, J. Biol. Chem. 279, 30546-30553.

4) Griffiths, J. S., Cheriyan, M., Corbell, J. B., Pocivavsek, L., Fierke, C. A. and Toone, E. J. (2004) A Bacterial Selection for the Directed Evolution of 2-Keto-3-deoxy-6-phosphogluconate aldolases, Bioorg. Med. Chem. 12, 4067-74.

5) Day-Storms, J. J., Niranjanakumari, S. and Fierke, C. A. (2004) Ionic Interactions between PRNA and P Protein in Bacillus subtilis RNase P Characterized using a Magnetocapture-Based Assay, RNA 10, 1595 - 1608.

6) Hernick, M., Gennadios, H. A., Whittington, D. A., Rusche, K. M., Christianson, D. W. and Fierke, C. A. (2005) UDP-3-O-(R-3-Hydroxymyristoyl)-N-Acetylglucosamine Deacetylase Functions Through a General Acid-Base Catalyst Pair Mechanism, J. Biol. Chem. 280, 16969-78.

7) Xiao, S., Day-Storms, J. J., Srisawat, C., Fierke, C. A. and Engelke, D. (2005) Characterization of Conserved Sequence Elements in Eukaryotic RNase P RNA Reveals Roles in Holoenzyme Assembly and tRNA Processing, RNA 11, 885-96.

8) Reigard, S. A., Zahn, T. J., Haworth, K. B., Hicks, K. A., Fierke, C. A. and Gibbs, R. A. (2005) Interplay of Isoprenoid and Peptide Substrate Specificity in Protein Farnesyltransferase, Biochemistry 44, 11214-23.

9) Rueda, D., Hsieh, J., Day-Storms, J. J., Fierke, C. A. and Walter, N. G. (2005) The 5' Leader of Precursor tRNAAsp Bound to the Bacillus subtilis RNase P Holoenzyme Has an Extended Conformation, Biochemistry 44, 16130-16139.

10) Pais, J. E., Bowers, K. E., Stoddard, A. K. and Fierke, C. A. (2005) A Continuous, Fluorescent Assay for Protein Prenyltransferases Measuring Diphosphate Release, Anal. Biochem. 345, 302-311.

11) Hartman, H. L., Hicks, K. A. and Fierke, C. A. (2005) Peptide Specificity of Protein Prenyltransferases is Determined Mainly by Reactivity Rather than Binding Affinity, Biochemistry 44, 15314-15324.

12) Hicks, K. A., Hartman, H. L. and Fierke, C. A. (2005) Upstream Polybasic region in Peptides Enhances Dual Specificity for Prenylation by both Protein Farnesyltransferase and Protein Geranylgeranyltransferase Type I, Biochemistry 44, 15325-15333.

13) Sumner, J. P., Westerberg, N. M., Stoddard, A. K., Hurst, T. K., Cramer, M., Thompson, R. B., Fierke, C. A. and Kopelman, R. (2006) DsRed as a Highly Sensitive, Selective and Reversible Fluorescence-Based Bioensor for Both Cu+ and Cu2+ Ions, Biosens. Bioelect. 15, 1302-1208.

14) Bozym, R. A., Thompson, R. B., Stoddard, A. K. and Fierke, C. A. (2006) Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor, ACS Chemical Biology, in press.

15) Fullerton, S. W. B., Griffiths, J. S., Merkel, A. B., Cheriyan, M., Wymer, N. J., Hutchins, M. J., Fierke, C. A., Toone, E. J. and Naismaith, J. H. (2006) Mechanism of the Class I KDPG aldolases, Bioorg. Med. Chem. Lett., in press.

Top


Biophysics Research Division University of Michigan 4028 Chemistry Building 930 North University Avenue Ann Arbor, MI 48109-1055 UMsitemaker	Phone: (734) 763-6722 Fax: (734) 764-3323 E-mail: biophysics@umich.edu Last Updated: 1/23/2006 © Copyright 2006 University of Michigan