Development of Multipurpose Probes and Affinity Reagents for Rapid Isolation and Visualization of Protein Complexes
Sponsor: DOE-Office of Biological and Environmental Research Genomics: Genomes to Life Program
Contact: Uljana Mayer-Cumblidge
Affinity isolation of intact protein complex (top) and visual depiction of identified proteins associated with the RNA polymerase complex (bottom) using newly developed multiuse affinity probes (MAPs). Individual columns represent the confidence level for five separate experiments for eluted protein using the control beads and RpoA bait. Identified gene accession numbers and common protein names are shown to the right and core enzyme components of RNA polymerase complex are highlighted in yellow. Scale associated with color choices for protein abundances is shown at the bottom of the figure, where white represents an Xcorr above 250. Of the 50 identified proteins, 26 are significantly above background (designated with asterisk and in bold). Enlarged View
Our long-term goal is to develop high-throughput methods for the rapid and quantitative characterization of protein complexes in microbial cells. The initial focus is on Shewanella oneidensis MR-1, whose metabolism is important in understanding both microbial energy production and environmental remediation. However, these strategies will be applicable to a wide range of microorganisms and will permit the identification of environmental conditions that affect the expression of critical proteins required for the formation of adaptive protein complexes that facilitate bacterial growth.
Our hypothesis is that identifying dynamic changes in these adaptive protein complexes will provide important insights into the metabolic regulatory strategies used by these organisms to adapt to environmental changes. We are implementing a strategy focusing on the development of multiuse protein probes 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 probe, 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 transient or low-affinity binding interactions in protein complexes is possible by engineering protein crosslinkers onto the bisarsenical affinity reagents. Furthermore, these same protein probes 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 that provides a direct measurement of the metabolic flow through defined biochemical pathways in response to environmental conditions.
Ultimately, these methods will permit an optimization of useful signaling and metabolic pathways to fulfill DOE goals involving efficient energy utilization, carbon sequestration, and environmental remediation. To accomplish these goals, we have three specific aims: 1) Identify multipurpose probes with optimized sequences for differential labeling using cell permeable orthogonal fluorescent probes, 2) Express tagged proteins and optimize affinity labeling in S. oneidensis MR-1, and 3) Develop improved affinity reagents with optional photocrosslinking extensions for in vivo stabilization and identification of protein complexes.