Cellular protein-protein interactions can be highly interconnected. Because of this complexity, it is often difficult to extract quantitative information on how each interaction contributes to distinct or overlapping cellular functions, and, moreover, how changes to individual interactions result in altered function or disease. We are developing an experimental platform for studying perturbations to multi-functional network “hub” proteins by combining high-throughput in vivo genetic interaction screening technology (Epistatic MiniArray Profile (E-MAP)) with mass-spectrometry and biophysical assays. Our case study protein is the highly-conserved multi-functional Gsp1/Ran GTPase switch that controls key eukaryotic processes. The approach first engineers defined perturbations to Gsp1/Ran protein-protein interactions by amino acid point mutations (“edge perturbations”). The second step determines the functional effects of these perturbations at the cellular and organism level in the model S. cerevisiae. We find that E-MAPs have a resolution that enables us to identify quantitative functional differences in vivo between individual point mutations, even those between different amino acid substitutions of the same residue. Our analysis reveals several classes of observed phenotypes that could be explained by the underlying biophysical perturbations of the on/off balance of the fundamental GTPase switch and considerable allosteric effects in the system.