Areas of Interest
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. We are particularly focussed on the mechanism of medically important metalloenzymes. We are investigating the catalytic mechanism and specificity of protein farnesyltransferase and protein geranylgeranyltransferase I. These enzymes catalyze the addition of a prenyl group onto a C-terminal cysteine on a variety of substrates involved in signal transduction. Compounds that inhibit FTase are being investigated as possible antitumor agents. A second enzyme, UDP-3-O-acyl-GlcNAC deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the pathway to form Lipid A, a crucial component of the outer membrane of gram negative bacteria. Inhibitors of this enzyme have antibacterial activity. To further the development of novel inhibitors we are elucidating detailed structure-function relationships in the active site of these proteins using mutagenesis, kinetic analysis, X-ray crystallography, and spectroscopic studies. Additionally, we have begun mechanistic studies of two related enzymes, histone deacetylase and protein palmitoyltransferase. For all of the metalloenzymes, a key question is the identify of the in vivo metal ion and whether metal switching is an important regulatory mechanism. Finally, we are developing methods for high-throughput screens of protein-bound transition metal ions for use in assaying all of the proteins in the yeast proteome to identify the yeast "metallome".
We also investigating the catalytic modes of ribozymes compared to proteins by determining the structure and mechanism of ribonuclease P (RNase P), a ribonucleoprotein complex that catalyzes the cleavage of tRNA precursors. We have demonstrated that the protein component enhances the catalytic efficiency by interacting with both P RNA and pre-tRNA. In the future, we will elucidate the structure of the holoenzyme using fluorescence resonance energy transfer, crystallography and spectroscopy. Finally, we will investigate the mechanism of yeast RNase P (in collaboration with Dr. D. Engelke) which contains one RNA and multiple protein subunits and purify, clone and characterize the RNase P from mammalian mitochondria which is proposed to be a protein catalyst.
Zinc, iron and copper ions are proposed to play important biological roles, especially in neurobiology, as well as playing important roles in the development of a number of diseases, including diabetes. Furthermore, a number of metals, such as lead and cadmium, are toxic. We are investigating the mechanisms of metal homeostasis and metal toxicity using a combination of biochemistry, genetics and imaging. To this end, we are redesigning the zinc metalloenzyme, carbonic anhydrase II, to optimize a fluorescent biosensor for measuring and imaging "readily exchangeable" metal ions in complex biological mixtures, such as cells, plasma and sea water and are developing similar sensors to measure cellular iron concentrations. Additionally, we are using X-ray fluorescence microprobe imaging to image total metal ions in wild-type and mutant yeast cells. These imaging methods are being used to understand basic mechanisms of metal homeostasis. Finally, we are examining the metal content and the mechanisms of metal insertion into proteins in vivo using biochemistry and analysis of libraries of deletion mutants. These studies should lead to a better understanding of the functions and regulation of biological transition metals.
Honors & Awards
1992-1997 Am. Heart Assoc. Established Investigator Award
2001 Faculty Recognition Award
2003 Jerome and Isabella Karle Collegiate Prof. of Chemistry
2005 Sarah Power Goddard Award
2005 Distinguished Faculty Achievement Award
2007 AAAS Fellow
2012 Repligen Award
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. 21, 1302-1208.
Sumner, J. P., Westerberg, N. M., Stoddard, A. K., Fierke, C. A. and Kopelman, R. (2006) Cu+ and Cu2+ Sensitive PEBBLE Fluorescent Nanosensors using DsRed as the Recognition Element, Sensors and Actuators B: Chemical 113, 760-767.
Frederickson, C. J., Giblin, L. J., Krezel, A., McAdoo, D. J., Mueller, R. A., Zeng, Y., Balaji, R. V., Masalha, R., Thompson, R. B., Fierke, C. A., Sarvey, J. M, de Valdenebro, M., Prough, D. S., Zornow, M. H., (2006) Concentrations of extracellular free zinc (pZn)e in the central nervous system during simple anesthetization, ischemia and reperfusion, Exp. Neurol. 198, 285-93.
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 1, 103-111.
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 Naismith, J. H. (2006) Mechanism of the Class I KDPG aldolases, Bioorg. Med. Chem. 14, 3002-3010.
Xiao, S., Hsieh, J., Nugent, R. L., Coughlin, D. J., Fierke, C. A. and Engelke, D. R. (2006) Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP, RNA 12, 1023-37.
Gantt, S. L., Gattis, S. G. and Fierke, C. A. (2006) Catalytic Activity and Inhibition of Human Histone Deacetylase 8 Is Dependent on the Identity of the Active Site Metal Ion, Biochemistry 45, 6170-8.
Johnson, K. M., Cleary, J., Fierke, C. A., Opipari, Jr, A. W. and Glick, G. D. (2006) Mechanistic Basis for therapeutic targeting of the mitochondrial F1F0-ATPase, ACS Chemical Biology 1, 304-308.
Gennadios, H. A., Whittington, D. A., Li, X., Fierke, C. A. and Christianson, D. W. (2006) Mechanistic Inferences from the Binding of Ligands to LpxC, a Zinc-Dependent Deacetylase, Biochemistry 45, 7940-8.
Hernick, M. and Fierke, C. A. (2006) Catalytic mechanism and molecular recognition of E. coli UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase probed by mutagenesis, Biochemistry 45, 15240-8.
Hernick, M. and Fierke, C. A. (2006) Molecular recognition by E. coli UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase is modulated by bound metal ions, Biochemistry 45, 14573-81.
Pais, J. E., Bowers, K. E. and Fierke, C. A. (2006) Measurement of the Î±-Secondary Kinetic Isotope Effect for the Reaction Catalyzed by Mammalian Protein Farnesyltransferase, J. Am. Chem. Soc. 128, 15086-7.
Getz, M. M., Andrews, A. J., Fierke, C. A. and Al-Hashimi, H. M. (2007) Structural Plasticity and Mg2+ Binding Properties of RNase P P4 from Combined Analysis of NMR Residual Dipolar Couplings and Motionally Decoupled Spin Relaxation, RNA 13, 251-66.
Niranjanakumari, S., Day-Storms, J. J., Ahmed, M., Hsieh, J., Zahler, N. H., Venters, R. A. and Fierke, C. A. (2007) Probing the Architecture of the B. subtilis RNase P Holoenzyme Active Site by Crosslinking and Affinity Cleavage, RNA 13, 521-535.
Krzysiak, A.J., Rawat, D.S., Scott, S.A., Pais, J.E., Handley, M., Harrison, M.L., Fierke, C.A., and Gibbs, R. A. (2007) Combinatorial Modulation of Protein Prenylation, ACS Chemical Biology 2, 385-389
Inamdar, M. V., Lastoskie, C. M., Fierke, C. A. and Sastry, A. M. (2007) A mobile trap algorithm for zinc detection using protein sensors, J. Chem. Phys. 127, 185102.
Cheriyan, M., Toone, E. J. and Fierke, C. A. (2007) Mutagenesis of the phosphate binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates, Protein Sci. 16, 2368-77.
Krzysiak, A. J., Scott, S. A., Hicks, K. A., Fierke, C. A. and Gibbs, R. A. (2007) Evaluation of Protein Farnesyltransferase Substrate Specificity using Synthetic Peptide Libraries, Bioor. Med. Chem. Lett. 17, 5548-51.
Walters, M.J., Srikannathasan, V., McEwan, A. R., Naismith, J. H., Fierke, C. A. and Toone, E. J. (2008) Characterization and Crystal Structure of Escherichia coli KDPGal Aldolase, Bioor. Med. Chem. 16, 710-20. http://www.ncbi.nlm.nih.gov/pubmed/17981470
Lipton, A. S., Heck, R. W., Hernick, M., Fierke, C. A. and Ellis, P. D. (2008) Residue Ionization in LpxC Directly Observed by 67Zn NMR Spectroscopy, J. Am. Chem. Soc 130,12671-9. http://www.ncbi.nlm.nih.gov/pubmed/18761443
Mason, A.B., Judson, G.L., Bravo, M.C., Edelstein, A., Byrne, S.L., James, N.G., Roush, E.D, Fierke, C.A., Bobst, C.E., Kaltashov, I.A. and Daughtery, M.A. (2008) Evolution reversed: The ability to bind iron restored to the N-lobe of murine inhibitor of carbonic anhydrase by strategic mutagenesis, Biochemistry 47, 9847-55. http://www.ncbi.nlm.nih.gov/pubmed/18712936
Hsieh, J., Walker, S.C., Fierke, C.A. and Engelke, D. R. (2009) Pre-tRNA Turnover Catalyzed by the Yeast Nuclear RNase P Holoenzyme is Limited by Product Release, RNA, in press. http://www.ncbi.nlm.nih.gov/pubmed/19095620
Dowling, D.P., Gantt, S.L., Gattis, S.G., Fierke, C.A., and Christianson, D.W. (2008) Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors, Biochemistry 47, 13554-63. http://www.ncbi.nlm.nih.gov/pubmed/19053282
Hougland, J.L., Scott, S.A., Gibbs, R. A. and Fierke, C.A. (2009) Context-dependent substrate recognition by protein farnesyltransferase, Biochemistry, in press.
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