This is the first continuation of an imaginative program of research aimed at using trapping methods to isolate reaction intermediates, and then study their structures using either monochromatic X-rays or Laue crystallography, as appropriate. The first project period led to a number of successful experiments, both in terms of method development and also in terms of actual intermediates that were studied. Building on this valuable experience, Dr. Stoddard now proposes to move in three directions: (i) to determine the structure of a cleaved hammerhead ribozyme structure, (ii) to see if the ribozyme cleavage reaction can be studied in time (iii) to determine the structures of two members of a novel class of endo nucleases, the intron coded enzymes CreI and PpoI. After structure determination by MIR, these enzymes will then be utilized in studies aimed at determining the structures of reaction intermediates. (iv) Dr. Stoddard aims to develop solvent trapping methods to study chemically labile intermediates. Specifically, he plans to study the reaction of the Michaelis complex of the superoxide anion and superoxide dismutase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM049857-05
Application #
2701597
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1994-05-01
Project End
2001-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
Shen, Betty W; Lambert, Abigail; Walker, Bradley C et al. (2016) The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease. J Mol Biol 428:206-220
Ho, Joanne M; Reynolds, Noah M; Rivera, Keith et al. (2016) Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli. ACS Synth Biol 5:163-71
Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill et al. (2015) Rational design of ?-helical tandem repeat proteins with closed architectures. Nature 528:585-8
Takeuchi, Ryo; Choi, Michael; Stoddard, Barry L (2015) Engineering of customized meganucleases via in vitro compartmentalization and in cellulo optimization. Methods Mol Biol 1239:105-32
Mandell, Daniel J; Lajoie, Marc J; Mee, Michael T et al. (2015) Biocontainment of genetically modified organisms by synthetic protein design. Nature 518:55-60
Park, Keunwan; Shen, Betty W; Parmeggiani, Fabio et al. (2015) Control of repeat-protein curvature by computational protein design. Nat Struct Mol Biol 22:167-74
Procko, Erik; Berguig, Geoffrey Y; Shen, Betty W et al. (2014) A computationally designed inhibitor of an Epstein-Barr viral Bcl-2 protein induces apoptosis in infected cells. Cell 157:1644-1656
Takeuchi, Ryo; Choi, Michael; Stoddard, Barry L (2014) Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization. Proc Natl Acad Sci U S A 111:4061-6
Stoddard, Barry L (2014) Homing endonucleases from mobile group I introns: discovery to genome engineering. Mob DNA 5:7
Zhao, Lei; Stoddard, Barry L (2014) Rapid determination of homing endonuclease DNA binding specificity profile. Methods Mol Biol 1123:127-34

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