Protein molecular switches functionally couple external signals (such as ligand binding) to functionality. Molecular switches have a wide variety of potential health related applications including the regulation of gene transcription, the modulation of cell signaling pathways, targeted drug delivery, drug transport, the creation of conditionally active toxic proteins, and the creation of molecular biosensors. Despite their great potential, the creation of protein switches has not been extensively explored, in part due to the paucity of general strategies for their engineering. Using combinatorial methods that integrate biological, chemical and engineering approaches, molecular switches will be engineered by a novel strategy called 'combinatorial domain insertion' using model proteins in 'proof-of-principle' experiments. In combinatorial domain insertion, two genes are fused such that one is randomly inserted within the other. In the model system chosen, Gene A codes for a binding protein that undergoes a conformational change in the presence of a signal (e.g. ligand binding). Gene B codes for a protein to be controlled (e.g. an enzyme). From these libraries, fusion proteins will be identified that functionally couple the two domains' functions (e.g. ligand binding modulates the enzyme's activity). The functional coupling is hypothesized to result from ligand-dependent conformational/stability changes in protein A that affect the activity of protein B. Representative switches obtained will be kinetically and structurally characterized. Through the systematic analysis afforded by a combinatorial approach and the biochemical and structural characterization of the switches created, models of the mechanism of switching will be developed and tested experimentally with the goal of elucidating general) principles that can be applied to the creation of molecular switches for biomedical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM066972-01A1
Application #
6684914
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
2003-07-01
Project End
2008-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
1
Fiscal Year
2003
Total Cost
$267,319
Indirect Cost
Name
Johns Hopkins University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Shelat, Nirav Y; Parhi, Sidhartha; Ostermeier, Marc (2017) Development of a cancer-marker activated enzymatic switch from the herpes simplex virus thymidine kinase. Protein Eng Des Sel 30:95-103
Choi, Jay H; Xiong, Tina; Ostermeier, Marc (2016) The interplay between effector binding and allostery in an engineered protein switch. Protein Sci 25:1605-16
Choi, Jay H; Zayats, Maya; Searson, Peter C et al. (2016) Electrochemical activation of engineered protein switches. Biotechnol Bioeng 113:453-6
Nicholes, N; Date, A; Beaujean, P et al. (2016) Modular protein switches derived from antibody mimetic proteins. Protein Eng Des Sel 29:77-85
Ribeiro, Lucas Ferreira; Tullman, Jennifer; Nicholes, Nathan et al. (2016) A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination. Biotechnol Biofuels 9:119
Shelat, Nirav Y; Parhi, Sidhartha; Ostermeier, Marc (2016) A Positive Selection for Nucleoside Kinases in E. coli. PLoS One 11:e0162921
Choi, Jay H; Ostermeier, Marc (2015) Rational design of a fusion protein to exhibit disulfide-mediated logic gate behavior. ACS Synth Biol 4:400-6
Choi, Jay H; Laurent, Abigail H; Hilser, Vincent J et al. (2015) Design of protein switches based on an ensemble model of allostery. Nat Commun 6:6968
Wright, R Clay; Khakhar, Arjun; Eshleman, James R et al. (2014) Advancements in the development of HIF-1?-activated protein switches for use in enzyme prodrug therapy. PLoS One 9:e114032
Chaikind, Brian; Ostermeier, Marc (2014) Directed evolution of improved zinc finger methyltransferases. PLoS One 9:e96931

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