Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Protein Production and Molecular Tags
103
Development and Application of Multipurpose Affinity Probes to Isolate Intact Protein Complexes Associated with Metal Reduction from Shewanella oneidensis MR-1
Liang Shi*, Thomas C. Squier* (thomas.squier@pnl.gov), M. Uljana Mayer*, Haishi Cao, Baowei Chen, Yuri A. Gorby, David F. Lowry, Jeff Mclean, Seema Verma, and Ping Yan
Pacific Northwest National Laboratory, Richland, WA
Our long-term goal is to develop high-throughput methods for the rapid isolation of intact protein complexes and validation of these complexes in living cells. This methodology utilizes a small genetically encoded protein tag with an 8 amino-acid sequence containing a tetracysteine motif, which can be captured using affinity reagents or labeled with fluorescent dyes in vivo to permit cellular validation of protein complexes. An important advantage of this strategy is that a single small and nonperturbing tag can be sequentially used to 1) isolate the intact protein complex for identification and structural analysis and 2) visualization of the location and abundance of the protein complex within cells (Chen et al., 2004; Mayer et al., 2005). Furthermore, by varying the architecture of the affinity reagent, multiple colors and photoactivatable cross-linkers can be incorporated into the design strategy to permit measurements of binding interactions within cells and the stabilization of transient interactions associated with signaling complexes.
Proof of principle for this approach has been achieved through the isolation of two protein complexes (i.e., RNA polymerase and the metal reductase complex) from S. 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 transient 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.
RNA polymerase is a well studied system, which contains a core complex containing RNA polymerase alpha2betabeta’ subunits as well as regulatory proteins associated with the differential regulation of transcription. Following the expression of a tagged subunit of the RNA polymerase core complex in S. oneidensis MR-1, we have isolated this complex using the synthesized affinity reagent immobilized on a glass bead (Mayer et al., 2005). A critical advantage of this method is the ability to release the intact complex using a mild, one-step procedure with a competing dithiol. In addition to the identification of the core subunit complex, additional regulatory factors were identified, including the universal stress protein.
To investigate whether the current approach will also permit the identification of membrane protein complexes, we have tagged genes identified by the Shewanella Federation to be involved in metal reduction, and isolated members of this important protein complex. In one experiment, the metal reductase MtrC [a decaheme c-type cytochrome tentatively identified as an outer membrane protein whose activity is required for efficient reduction of Mn(IV) and Fe(III)] was genetically tagged and used to isolate two high-affinity heme-containing binding subunits in the complex that were not previously identified (i.e., OmcA and MtrA). The isolated MtrC complex maintained its activity to reduce Fe(III). This Fe(III)-reducing activity was enhanced by addition of purified MtrA, even though purified MtrA itself possessed no Fe(III)-reducing activity. Validation of this protein complex was achieved following purification of the individual proteins, using the affinity reagent dyes to measure the structural interactions between these proteins.
In summary, these multiuse affinity reagents have the advantage over other affinity tags for the high-throughput identification of protein binding partners, in that 1) the small tag can be rapidly cloned into the protein of interest and leads to minimal perturbations of binding interactions, 2) proteins are not denatured following elution permitting purification of the intact complex that can thus be further validated and studied by structural methods, and 3) the affinity reagents are cell permeable and can be used for imaging measurements to monitor protein-protein interactions in live cells.
References- Chen, B., M. U. Mayer, L. M. Markille, D. L. Stenoien, and T. C. Squier (2004) Dynamic motion of helix A in the amino-terminal domain of calmodulin is stabilized upon calcium activation. Biochemistry, in press.
- Mayer, M. U., L. Shi, and T. C. Squier (2005) One-step, non-denaturing isolation of an RNA Polymerase core enzyme complex using an improved multi-affinity probe resin. J. Am. Chem. Soc., submitted.
* Presenting author
