Development of Multipurpose Tags and Affinity Reagents for Rapid Isolation and Visualization of Protein Complexes
EMSL Project ID
7195c
Abstract
Our long-term goal is to develop high-throughput methods for the rapid and quantitative characterization of protein complexes in microbial cells. We propose to implement a strategy focusing on the development of multiuse protein tags engineered around a tetracysteine motif (i.e., CCXXCC), which has previously been shown to provide a highly selective binding site for cell permeable arsenic-containing affinity reagents that can be used to first identify and then validate protein complexes in living cells. Taking advantage of the large increase in the fluorescence signal associated with binding the proposed fluorescent affinity reagents to the protein tag, it will be possible to use on-line detection to monitor affinity isolation of protein complexes and rapidly identify the proteins in the complex using mass spectrometry. Identification of low-affinity binding interactions in protein complexes is possible by engineering protein crosslinkers onto the biarsenical affinity reagents. Furthermore, these same protein tags and affinity reagents will permit real-time visualization of steady-state protein abundance and protein-protein interactions, permitting validation of identified protein complexes under cellular conditions and the high-throughput identification of metabolic flow through defined biochemical pathways in response to environmental conditions. Ultimately, these methods will permit an optimization of useful metabolic pathways to fulfill Department of Energy (DOE) goals involving efficient energy utilization, carbon sequestration, and environmental remediation. To accomplish these goals, we propose several specific aims, but with respect to this user proposal, only one aim requires NMR: Identify multipurpose tags with optimized sequences for differential labeling using cell permeable orthogonal fluorescent probes.
Project Details
Project type
Capability Research
Start Date
2005-12-20
End Date
2006-04-24
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
A Red Cy3-Based Biarsenical Fluorescent Probe Targeted to a Complementary Binding Peptide Haishi Cao, Yijia Xiong, Ting Wang, Baowei Chen, Thomas C. Squier, and M. Uljana Mayer- Cell Biology and Biochemistry Group, Pacific Northwest National Laboratory, Richland, Washington 99352 Received January 1, 2007; E-mail: uljana.mayer@pnl.gov
CrAsH: a biarsenical multi-use affinity probe with low non-specific fluorescence{ Haishi Cao, Baowei Chen, Thomas C. Squier and M. Uljana Mayer- Received (in Cambridge, MA, USA) 24th February 2006, Accepted 10th May 2006 First published as an Advance Article on the web 17th May 2006 DOI: 10.1039/b602699k
Verma S, Xiong Y, Mayer MU, Squier TC (2007) Remodeling of the Bacterial RNA Polymerase Supramolecular Complex in Response to Environmental Conditions. Biochemistry 46: 3023-3035
Yan P, Y Xiong, B Chen, S Negash, TC Squier, and MUljana Mayer. 2006. "Fluorophore-Assisted Light Inactivation of Calmodulin Involves Singlet-Oxygen Mediated Cross-Linking and Methionine Oxidation." Biochemistry 45(15):4736-4748.