Dr. Barry L. Stoddard

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Fred Hutchinson Cancer Research Center
Basic Sciences Division
Program in Structural Biology
Full Member
University of Washington
School of Medicine
Biochemistry
Affiliate Professor
Professional Headshot of Barry L. Stoddard

Mailing Address

Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N.
A3-025
Seattle, Washington 98109-1024
United States

Contact Information

Phone: (206) 667-4031
Fax: (206) 667-3331
bstoddar@fhcrc.org
http://www.fhcrc.org/science/labs/stoddard/index.html

Qualifications

Ph.D., Massachusetts Institute of Technology, Biophysical Chemistry, 1990.
B.A., Whitman College, Chemistry, 1985.

Expertise and Research Interests

The goal of this laboratory is to understand the structure/function relationships of several interesting biological systems at the atomic level. The tools employed are X-ray crystallography, computer modelling, and genetic manipulation of the molecules of interest. Three projects are summarized below:

PROJECT 1: Structure, function and engineering of intron-encoded protein factors.

Intron-encoded proteins ('homing endonucleases'), found in eubacteria, archea, and single cell eukaryotes, recognize DNA sequences with very high specificity and promote the gene-specific transfer of introns and inteins within host genomes. In addition, many of these proteins also act as specific cofactors for intron-splicing (displaying 'maturase' activity), by forming tightly bound ribonucleoprotein complexes to their cognate introns.

Over the past five years, we have determined the structure and mechanism of several naturally occuring homing endonucleases. More recently, we have begun to address the problem of re-engineering these proteins to recognize DNA target sequences of our own choice and design. The development of single-protein gene-specific reagents (SP-GSRs) would be a major step towards the creation of artificial biomolecules that could be used as diagnostic reagents for genetic disorders, sensors against bacterial and viral pathogens, and research tools for molecular and cellular biology. An SP-GSR is a protein molecule which can recognize and bind a single unique target site located within an entire complex genome. The protein could also be engineered to carry out a variety of chemical processes at the DNA target site. Such a molecule would have to be extraordinarily specific, because the size of the human genome is quite large--approximately four billion base pairs in length. An arsenal of such molecules, directed against DNA sequences of an investigator's own choosing, does not currently exist.

We have initiated a project to combine genetic, biochemical and computational experiments in order to design and engineer artifical SP-GSRs from homing endonucleases. It should be possible to create new DNA specificities for these proteins by combining genetic selection strategies with computational protein redesign methods. New methods of computational molecular design and of genetic selection must be invented and then used in a rational design cycle for this project to succeed.

PROJECT 2: Structure, function and engineering of nucleotide synthesis and salvage enzymes for directed anticancer gene therapy.

Nucleotide synthesis and salvage enzymes are well-documented targets for the design of inhibitors for chemotherapeutic and antibiotic drug development, and for the creation of altered enzymatic catalysts for gene-therapy applications. Our lab is working on structural and mechanistic studies of several enzymes from this broad metabolic area, including tetrahydrofolate synthetase, cyclohydrolase and dehydrogenase, cytosine deaminase (CD), deoxycytidine kinase (dCK) and human ribonucleotide reductase. In particular, the selection and engineering of CD and dCK variants that act efficiently on nucleotide analogues is an important goal, for the purpose of creating enhanced catalysts for anticancer prodrug-gene therapy.

Prodrug gene therapy is a therapeutic strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme which is capable of converting a nontoxic prodrug into a potent cytotoxin. Such a method allows selective eradication of tumor cells while sparing normal tissue from significant cell killing. The effectiveness of this strategy is dependent on a bystander effect in which untransfected tumor cells are killed through active or passive transport of the cytotoxic enzyme product. Several enzyme/prodrug combinations are under active investigation, demonstrating effectiveness in both tissue culture and animal models. However, the combination of low transfection efficiencies and poor turnover of prodrug substrates limit the efficiency of cell killing in the tumor. In order to improve such therapies, enzyme variants must be selected and engineered for enhanced turnover of the prodrug substrate. In this project, a collaboration of two laboratories are optimizing cytosine deaminase and deoxycytidine kinase for prodrug suicide gene therapy, using a combination of structural biology and directed evolution screens, and to test the efficacy of enzyme variants in cell line and animal models.

PROJECT 3: Protein Engineering (in collaboration with Drs. David Baker and Ray Monnat, Jr. at the University of Washington and Dr. Andrew Scharenberg at the Childrens Hospital Research Institute in Seattle).

As described in projects 1 and 2 above, a major focus of our research is the selection and engineering of a variety of enzyme catalysts for altered substrate specificities, and for altered biophysical properties such as enhanced stability and catalytic lifetimes. In 2002, we carried out what was (for us) a seminal experiment in which we created a fully active, chimeric homing endonuclease capable of recognizing and cleaving a complementary chimeric target site (article (3) below). As part of that project, we discovered that computational algorithms being developed by the laboratory of David Baker at the UW could be successfully applied to such a problem, by repacking a protein interface and creating a novel domain packing architecture. Subsequently, we have used similar algorithms with great success for a variety of purposes, such as the thermostabilization of an enzyme catalyst for prodrug gene therapy.

Our current and long-term aims is to continue to develop and exploit computational engineering algorithms to alter substrate binding specificities for homing endonucleases and cytosine deaminase enzymes. Both projects involve the repacking and optimization of protein-nucleic acid interfaces, which poses substantial challenges relative to protein cores and interfaces.

Other Expertise

Biotech advisory board, Western Washington University

Consultant for Payload Systems, Inc. 1987 through 1992: Protein Crystallization in a microgravity environment. Participated in an academic/industrial collaboration for these experiments on the Russian Space Station 'MIR'.

Member, scientific advisory board for NASA Project on Iterative Biological Crystallization (2001 - present)

Member, Defense/Science StudyGroup (DSSG) 2000-2001

Customer/science advisory board, Molecular Structure Corporation, 1999

Consultant, New England Biolabs and Biohelix (Ipswitch, MA) 2005 - present

Consultant and Member of Scientific Advisory Board, Targeted Growth, Inc (Seattle, WA) 2006 - present.

Keywords

COS Keywords:

Antibiotics, Biochemistry, Biophysics, Cell Biology, Chemical Kinetics, Chemotherapeutic Agents, Computer Modeling, Crystallography, Developmental Biology, Drug Design, Genetic Manipulation, Genetics, Human Physiology, Pharmacology, Structural Biology.

Additional Terms:

Active Site, Carbon Nitrogen Ligase, Chemical Cleavage, Chemical Kinetics, Chemical Model, Cofactor, Computer Simulation, Conformation, Endonuclease, Enzyme Complex, Enzyme Inhibitor, Enzyme Mechanism, Enzyme Structure, Enzyme Substrate Complex, Flash Photolysis, Homing Endonuclease, Isocitrate Dehydrogenase, Laue Diffraction, Ligand, Mutant, Nadh Phosphate, Nucleic Acid Structure, Ribozyme, Structural Biology, Tetrahydrofolate, Time Resolved Data, Tricarboxylate, X Ray Crystallography.

Languages

(Reading, Writing, Speaking)

English: (Fluent, Fluent, Fluent)

Memberships

American Association for the Advancement of Science
American Crystallographic Association

Previous Positions

1990-1992, Postdoctoral Research Fellow, University of California, Berkeley, College of Arts and Sciences, Molecular and Cellular Biology, Biochemistry

Publications

  • Stoddard BL, Nuclease enzymes, Methods (san Diego, Calif.), 28(3), 301, November 2002 Abstract
  • Chevalier BS, Kortemme T, Chadsey MS, Baker D, Monnat RJ, Stoddard BL, Design, activity, and structure of a highly specific artificial endonuclease, Molecular Cell, 10(4), 895-905, October 2002 Abstract
  • Galburt, E. A., Pelletier, J., Wilson, G. and Stoddard, B. L., Structure of a tRNA repair enzyme and molecular biology workhorse: T4 polynucleotide kinase, Structure, 10, 1249 - 1260, September 2002
  • Ireton GC, McDermott G, Black ME, Stoddard BL, The structure of Escherichia coli cytosine deaminase, Journal of Molecular Biology, 315(4), 687-97, January 2002 Abstract
  • Ireton GC, Black ME, Stoddard BL, Crystallization and preliminary X-ray analysis of bacterial cytosine deaminase, Acta Crystallographica, Section D: Biological Crystallography, 57(Pt 11), 1643-5, November 2001 Abstract
  • Chevalier BS, Stoddard BL, Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility, Nucleic Acids Research, 29(18), 3757-74, September 2001 Abstract
  • Spiegel PC Jr, Jacquemin M, Saint-Remy JM, Stoddard BL, Pratt KP, Structure of a factor VIII C2 domain-immunoglobulin G4kappa Fab complex: identification of an inhibitory antibody epitope on the surface of factor VIII, Blood, 98(1), 13-9, July 2001 Abstract
  • Stoddard BL, Trapping reaction intermediates in macromolecular crystals for structural analyses, Methods (san Diego, Calif.), 24(2), 125-38, June 2001 Abstract
  • Chevalier BS, Monnat RJ Jr, Stoddard BL, The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites, Nature Structural Biology, 8(4), 312-6, April 2001 Abstract
  • Bond CJ, Jurica MS, Mesecar A, Stoddard BL, Determinants of allosteric activation of yeast pyruvate kinase and identification of novel effectors using computational screening, Biochemistry, 39(50), 15333-43, December 2000 Abstract
  • Liu ML, Shen BW, Nakaya S, Pratt KP, Fujikawa K, Davie EW, Stoddard BL, Thompson AR, Hemophilic factor VIII C1- and C2-domain missense mutations and their modeling to the 1.5-angstrom human C2-domain crystal structure, Blood, 96(3), 979-87, August 2000 Abstract
  • Galburt EA, Chadsey MS, Jurica MS, Chevalier BS, Erho D, Tang W, Monnat RJ Jr, Stoddard BL, Conformational changes and cleavage by the homing endonuclease I-PpoI: a critical role for a leucine residue in the active site, Journal of Molecular Biology, 300(4), 877-87, July 2000 Abstract
  • Galburt EA, Stoddard BL, Restriction endonucleases: one of these things is not like the others [news, Nature Structural Biology, 7(2), 89-91, February 2000 Abstract
  • Galburt EA, Chevalier B, Tang W, Jurica MS, Flick KE, Monnat RJ Jr, Stoddard BL, A novel endonuclease mechanism directly visualized for I-PpoI, Nature Structural Biology, 6(12), 1096-9, December 1999 Abstract
  • Galburt EA, Chevalier B, Tang W, Jurica MS, Flick KE, Monnat RJ Jr, Stoddard BL, A novel endonuclease mechanism directly visualized for I-PpoI., Nat. Struct. Biol, 6(12), 1096-9, December 1999 Abstract
  • Pratt KP, Shen BW, Takeshima K, Davie EW, Fujikawa K, Stoddard BL, Structure of the C2 domain of human factor VIII at 1.5 A resolution, Nature, 402(6760), 439-42, November 1999 Abstract
  • Jurica MS, Stoddard BL, Homing endonucleases: structure, function and evolution., Cellular and Molecular Life Sciences, 55(10), 1304-26, 15 Aug 1999 Abstract
  • Shen BW, Dyer DH, Huang JY, D''Ari L, Rabinowitz J, Stoddard BL, The crystal structure of a bacterial, bifunctional 5,10 methylene-tetrahydrofolate dehydrogenase/cyclohydrolase., Protein Science, 8(6), 1342-9, June 1999 Abstract
  • Bryant MD, Flick KE, Koduri RS, Wilton DC, Stoddard BL, Gelb MH, 1,3-Dioxane-4,6-dione-5-carboxamide-based inhibitors of human group IIA phospholipase A: X-ray structure of the complex and interfacial selection of inhibitors from a structural library., Bioorganic and Medicinal Chemistry Letters, 9(8), 1097-102, 19 Apr 1999 Abstract
  • M. D. Bryant, K. E. Flick, R. S. Koduri, D. C. Wilton, B. L. Stoddard, and M. H. Gelb, 1,3-dioxane-4,6-dione-5-carboxamide-based inhibitors of human group IIA phospholipase A2: X-ray structure of the complex and interfacial selection of inhibitors from a structural library, Bioorganic and Medicinal Chemistry Letters, 9, 1097 - 1102, 1999
  • Stoddard, B.L., Cohen, B.E., Brubaker, M., Mesecar, A.D., Koshland, D. E. Jr., Millisecond Laue structures of an enzyme-product complex using photocaged substrate analogs, Nature Structural Biology, 5(10), 891 - 897, October 1998
  • Jurica, M.S., Monnat, R.J. Jr., Stoddard, B. L., DNA recognition and cleavage by the LAGLIDADG homing endonuclease I-CreI, Molecular Cell, 2, 469 - 476, October 1998
  • Stoddard BL, New results using Laue diffraction and time-resolved crystallography., Curr Opin Struct Biol, 8(5), 612-8, October 1998 Abstract
  • Jurica M S, Stoddard B L, Mind your B's and R's: bacterial chemotaxis, signal transduction and protein recognition., Structure, 6(7), 809-13, 15 Jul 1998 Abstract
  • Flick K E, Jurica M S, Monnat R J Jr, Stoddard B L, DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI., Nature, 394(6688), 96-101, 2 Jul 1998 Abstract
  • Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T, Stoddard BL, The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate., Structure, 6(2), 195-210, 15 1998 Abstract
  • Stoddard, B. L., New results using Laue diffraction and time-resolved crystallography, Current Opinions in Structural Biology, 8, 612 - 618, 1998
  • Stoddard B L, Pietrokovski S, Breaking up is hard to do, Nature Structural Biology, 5(1), 3-5, Jan 1998 Abstract
  • Flick K E, McHugh D, Heath J D, Stephens K M, Monnat R J Jr, Stoddard B L, Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum., Protein Science, 6(12), 2677-80, December 1997 Abstract
  • Cohen B E, Stoddard B L, Koshland D E Jr, Caged NADP and NAD. Synthesis and characterization of functionally distinct caged compounds., Biochemistry, 36(29), 9035-44, 22 Jul 1997 Abstract
  • Mesecar A D, Stoddard B L, Koshland D E Jr, Orbital steering in the catalytic power of enzymes: small structural changes with large catalytic consequences., Science, 277(5323), 202-6, 11 Jul 1997 Abstract
  • Heath P J, Stephens K M, Monnat R J Jr, Stoddard B L, The structure of I-Crel, a group I intron-encoded homing endonuclease, Nature Structural Biology, 4(6), 468-76, June 1997 Abstract
  • Heath PJ, Stephens KM, Monnat RJ Jr, Stoddard BL, The structure of I-Crel, a group I intron-encoded homing endonuclease [see comments, Nat. Struct. Biol, 4(6), 468-76, 1997 Abstract
  • Stephens K M, Monnat R J Jr, Heath P J, Stoddard B L, Crystallization and preliminary X-ray studies of I-CreI: a group I intron-encoded endonuclease from C. reinhardtii., Proteins, 28(1), 137-9, May 1997 Abstract
  • Ellman J, Stoddard B, Wells J, Combinatorial thinking in chemistry and biology., Proceedings of the National Academy of Sciences (USA), 94(7), 2779-82, 1 Apr 1997 Abstract
  • Cheung E, D'Ari L, Rabinowitz J C, Dyer D H, Huang J Y, Stoddard B L, Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli., Proteins, 27(2), 322-4, February 1997 Abstract
  • D'Ari L, Cheung E, Rabinowitz J C, Bolduc J M, Huang J Y, Stoddard B L, Purification, crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici., Proteins, 27(2), 319-21, February 1997 Abstract
  • Scott W G, Murray J B, Arnold J RP, Stoddard B L, Klug A, Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme., Science, 274(5295), 2065-9, 20 Dec 1996 Abstract
  • Stoddard B L, Flick K E, Calcineurin-immunosuppressor complexes., Current Opinion In Structural Biology, 6(6), 770-5, December 1996 Abstract
  • Stoddard B L, Caught in a chemical trap, Nature Structural Biology, 3(11), 907-9, November 1996 Abstract
  • Stoddard B L, Dean A, Bash P A, Combining Laue diffraction and molecular dynamics to study enzyme intermediates., Nature Structural Biology, 3(7), 590-5, July 1996 Abstract
  • Brubaker M J, Dyer D H, Stoddard B, Koshland D E Jr, Synthesis, kinetics, and structural studies of a photolabile caged isocitrate: a catalytic trigger for isocitrate dehydrogenase., Biochemistry, 35(9), 2854-64, 5 Mar 1996 Abstract
  • Stoddard B L, Intermediate trapping and laue X-ray diffraction: potential for enzyme mechanism, dynamics, and inhibitor screening., Pharmacology and Therapeutics, 70(3), 215-56, 1996 Abstract
  • Stoddard B L, Farber G K, Direct measurement of reactivity in the protein crystal by steady-state kinetic studies., Structure, 3(10), 991-6, 15 Oct 1995 Abstract
  • Bolduc J M, Dyer D H, Scott W G, Singer P, Sweet R M, Koshland D E Jr, Stoddard B L, Mutagenesis and Laue structures of enzyme intermediates: isocitrate dehydrogenase [published erratum appears in Science 1995 Oct 20;270(5235):365], Science, 268(5215), 1312-8, 2 Jun 1995 Abstract
  • Lee M E, Dyer D H, Klein O D, Bolduc J M, Stoddard B L, Koshland D E Jr, Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP(+)-dependent isocitrate dehydrogenase from Escherichia coli., Biochemistry, 34(1), 378-84, 10 Jan 1995 Abstract
  • Scott W G, Stoddard B L, Transmembrane signalling and the aspartate receptor., Structure, 2(9), 877-87, 15 Sep 1994 Abstract
  • Stoddard B L, Koshland D E Jr, Structure of isocitrate dehydrogenase with alpha-ketoglutarate at 2.7-A resolution: conformational changes induced by decarboxylation of isocitrate., Biochemistry, 32(36), 9317-22, 14 Sep 1993 Abstract
  • Stoddard B L, Dean A, Koshland D E Jr, Structure of isocitrate dehydrogenase with isocitrate, nicotinamide adenine dinucleotide phosphate, and calcium at 2.5-A resolution: a pseudo-Michaelis ternary complex., Biochemistry, 32(36), 9310-6, 14 Sep 1993 Abstract
  • Stoddard B L, Koshland D E Jr, Molecular recognition analyzed by docking simulations: the aspartate receptor and isocitrate dehydrogenase from Escherichia coli., Proceedings of the National Academy of Sciences (USA), 90(4), 1146-53, 15 Feb 1993 Abstract
  • Stoddard B L, Farber G K, Strong R K, The facts and fancy of microgravity protein crystallization., Biotechnology and Genetic Engineering Reviews, 11, 57-77, 1993 Abstract
  • Stoddard B L, Bui J D, Koshland D E Jr, Structure and dynamics of transmembrane signaling by the Escherichia coli aspartate receptor., Biochemistry, 31(48), 11978-83, 8 Dec 1992 Abstract
  • Stoddard B L, Strong R K, Arrott A, Farber G K, Mir for the crystallographers' money., Nature, 360(6402), 293-4, 26 Nov 1992 Abstract
  • Stoddard B L, Koshland D E Jr, Prediction of the structure of a receptor-protein complex using a binary docking method., Nature, 358(6389), 774-6, 27 Aug 1992 Abstract
  • Stoddard B L, Biemann H P, Koshland D E Jr, Receptors and transmembrane signaling., Cold Spring Harbor Symposia On Quantitative Biology, 57, 1-15, 1992 Abstract

Profile Details

Last Updated: 6/13/2007

COS Expertise ID #544557
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