K-130. Development of a Quantitative Method for Measuring Binding Affinities in Live Cells Using Fluorescence Imaging Techniques

A. N. Edwards1,2, J. D. Fowlkes2, R. F. Standaert2, M. J. Doktycz2, J. L. Morrell-Falvey2;
1Univ. of Tennessee, Knoxville, TN, 2Oak Ridge Natl. Lab., Oak Ridge, TN.

Characterizing protein interactions within cellular networks is essential for understanding biological function, organization, and mechanisms of regulation. Methods exist to assay protein interactions; however, none are known to provide both confirmation of protein interactions and simultaneous quantification of biophysical parameters (binding strengths and association/dissociation rates) in vivo. These measurements are important for understanding complex biological networks as it is well recognized that subtle changes in binding affinities can alter protein function. We are currently developing an approach that combines an imaging-based protein interaction assay with an inverse fluorescence photobleaching and recovery technique (iFRAP), and computer simulations to provide a facile, general method for quantifying protein binding affinities in vivo. Using the assay adapted from Ding et al. (J. Bact 184:5572), a positive protein interaction is revealed by simple fluorescence localization patterns in E. coli cells due to spatial confinement of the interaction pair at the cell pole, which makes this assay amenable to automated image analysis and high throughput screening. We routinely use this assay to confirm protein interactions that are identified from mass spectrometry-based analyses of affinity isolates as a part of the DOE’s Center for Molecular and Cellular Systems. In addition, spatial confinement of the interacting proteins should also enable measurements of binding constants in vivo using iFRAP. To demonstrate this novel measurement method, we have focused on obtaining quantitative data from well-characterized protein interactions, such as streptavidin-SBP and eukaryotic Importin α (Impα) with various nuclear localization signals (NLSs). Using these protein pairs, we have determined that the protein interaction assay is sensitive enough to detect Kd values ranging from 1 nM to 3000 nM. Initial FRAP studies were also applied to characterize GFP and GFP fusion protein diffusion in E. coli. Using these measurements, current iFRAP studies are focused on quantifying the binding affinity between NLSs and Impα.