This project will develop powerful new tools to determine when and where proteins are made in complex cellular systems. Reactive amino acid analogs will be incorporated into cellular proteins and selectively conjugated to probes for visualization, isolation and identification of newly synthesized proteins in both prokaryotic and eukaryotic cells. Most importantly, these approaches can provide both spatial and temporal resolution in analysis of cellular protein synthesis. Cell-selectivity is achieved by controlled expression of mutant aminoacyl-tRNA synthetases;in systems containing multiple cell types, amino acid labeling is confined to cells in which the mutant synthetase is active. This project will explore the use of such methods to elucidate the mechanisms by which bacteria evade the defenses of mammalian hosts, to examine the process of quorum sensing (which is essential to the expression of virulence by bacterial pathogens), and to interrogate protein synthesis in a cell-selective manner in the model organism Caenorhabditis elegans. These studies will establish powerful, general platforms for systems-level characterization of biological phenomena ranging from development to the treatment of disease.

Public Health Relevance

This project will provide new methods to elucidate the mechanisms by which bacteria attempt to evade the defenses of their mammalian hosts, to examine how bacteria communicate with one another to express virulence factors and form antibiotic-resistant biofilms, and to identify the different sets of proteins made by individual cells in living animals. These studies will establish new windows on biological phenomena ranging from growth and development to the treatment of infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062523-11
Application #
8502675
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Smith, Ward
Project Start
2001-01-01
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
11
Fiscal Year
2013
Total Cost
$340,637
Indirect Cost
$120,537
Name
California Institute of Technology
Department
Chemistry
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Bagert, John D; Xie, Yushu J; Sweredoski, Michael J et al. (2014) Quantitative, time-resolved proteomic analysis by combining bioorthogonal noncanonical amino acid tagging and pulsed stable isotope labeling by amino acids in cell culture. Mol Cell Proteomics 13:1352-8
Lu, Ying Y; Sweredoski, Michael J; Huss, David et al. (2014) Prometastatic GPCR CD97 is a direct target of tumor suppressor microRNA-126. ACS Chem Biol 9:334-8
Hatzenpichler, Roland; Scheller, Silvan; Tavormina, Patricia L et al. (2014) In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry. Environ Microbiol 16:2568-90
Van Deventer, James A; Yuet, Kai P; Yoo, Tae Hyeon et al. (2014) Cell surface display yields evolvable, clickable antibody fragments. Chembiochem 15:1777-81
Mahdavi, Alborz; Szychowski, Janek; Ngo, John T et al. (2014) Identification of secreted bacterial proteins by noncanonical amino acid tagging. Proc Natl Acad Sci U S A 111:433-8
Yuet, Kai P; Tirrell, David A (2014) Chemical tools for temporally and spatially resolved mass spectrometry-based proteomics. Ann Biomed Eng 42:299-311
Mahdavi, Alborz; Segall-Shapiro, Thomas H; Kou, Songzi et al. (2013) A genetically encoded and gate for cell-targeted metabolic labeling of proteins. J Am Chem Soc 135:2979-82
Ngo, John T; Schuman, Erin M; Tirrell, David A (2013) Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells. Proc Natl Acad Sci U S A 110:4992-7
Hinz, Flora I; Dieterich, Daniela C; Tirrell, David A et al. (2012) Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish. ACS Chem Neurosci 3:40-49
Van Deventer, James A; Fisk, John D; Tirrell, David A (2011) Homoisoleucine: a translationally active leucine surrogate of expanded hydrophobic surface area. Chembiochem 12:700-2

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