G proteins regulate intracellular signaling processes through their interactions with guanine nucleotides, GDP and GTP, and initiate numerous physiological responses. In normal conditions, G protein functions are regulated in a tightly controlled signaling network through complex formation with partner proteins, especially G protein‐coupled receptors (GPCRs). However, loss‐of‐function or gain‐of‐function mutations in the Gα subunit of G proteins disrupt this process, circumventing the usual control mechanisms and causing changes in G protein activity that result in serious illnesses. Our studies focused on the stimulatory class of trimeric G proteins, Gs, and mutations in Gαs associated with multiple cancers1. While the structure of one Gαs variant has been determined and additional Gαs variants have been characterized biochemically, there remains a need for an overall mechanistic framework for relating mutations in Gαs to changes in G protein signaling. To fill in this gap in knowledge, we investigated the interactions of Gαs variants with nucleotides using saturation transfer difference nuclear magnetic resonance (STD‐NMR) spectroscopy in aqueous solutions. In STD‐NMR experiments, we observed interactions between nucleotides and the Gαs variant proteins at atomic resolution, which provided information on nucleotide binding modes. STD‐NMR data of one Gαs variant closely correlated with literature data of the structure and activity of the same variant protein2, establishing a foundation for extending the analysis of NMR data to seven additional disease‐associated Gαs variant proteins for which no structures are available. These NMR data were combined with correlative biophysical data that measured the overall fold of the Gαs‐nucleotide complexes and with functional data reporting on the relative affinities of the Gαs variants for GDP and GTP. Integrating these data together provided a conceptual framework for categorizing variations in Gαs amino acid sequences with changes in signaling activity. Surprisingly, differences in the binding modes of the Gαs variants did not always directly correlate with their different functions, suggesting that mutations in Gαs that impact nucleotide exchange are nuanced and complex. These results imply that strategies for the development of small molecule drugs may need to be specifically tailored for each disease associated Gαs variant. References O’Hayre, M. et. al. The emerging mutational landscape of G proteins and G protein‐coupled receptors in cancer. Nat. Rev. Cancer. 13, 412‐424 (2013). Hu, Q. & Shokat, K. M. Disease‐Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP. Cell. 173, 1254–1264 (2018).