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FRET Screening of Protein-Protein Interactions in Shewanella (Orr GTL-Imaging, PNNL/ORNL Scope #43930)


EMSL Project ID
17691

Abstract

Fluorescence resonance energy transfer (FRET) imaging and spectroscopy techniques will be used to identify specific molecular interactions among proteins in Shewanella. The techniques incorporate approaches that identify protein interactions in the intact natural cellular environment. Relying on FRET to report distances below 100 Å, we will identify specific interactions between pairs of full-length cytoplasmic, inner membrane, and TAT secreted proteins that are fused to either, CFP or YFP, in the living cell. In addition, dye-labeled antibody fragments against specific proteins will be applied to permeabilized cells, to identify molecular interactions. FRET imaging and spectroscopy will be carried out using high-sensitivity cutting-edge technologies. These technologies include total internal reflection (TIRF) microscopy, high quantum yield and low noise CCD cameras and APDs, and advanced design of FRET optics. Together, these technologies allow quantitative analyses of FRET efficiency by precise pixel-by-pixel measurements of donor/acceptor emission ratios and donor fluorescence life-time within an image of a single bacterium, as well as spectroscopic bulk measurements for synchronized fast and transient interactions. Through this effort, specific protein-protein interactions in the bacterial cell will be unambiguously verified, including interactions among membrane-associated proteins.

Project Details

Project type
Exploratory Research
Start Date
2006-01-19
End Date
2006-11-08
Status
Closed

Team

Principal Investigator

Galya Orr
Institution
Environmental Molecular Sciences Laboratory

Team Members

David Panther
Institution
Pacific Northwest National Laboratory

Brian Hooker
Institution
Pacific Northwest National Laboratory

H Wiley
Institution
Environmental Molecular Sciences Laboratory

Margaret Romine
Institution
Pacific Northwest National Laboratory