Thermodynamic analysis of the GASright transmembrane motif supports energetic model of dimerization

The GASright motif, best known as the fold of the glycophorin A transmembrane dimer, is one of the most common dimerization motifs in membrane proteins, characterized by its hallmark GxxxG-like sequence motifs (GxxxG, AxxxG, GxxxS, and similar). Structurally, GASright displays a right-handed crossing angle and short interhelical distance. Contact between the helical backbones favors the formation of networks of weak hydrogen bonds between Cei-H carbon donors and carbonyl accep-tors on opposing helices (Cei-H center dot center dot center dot O1/4C). To understand the factors that modulate the stability of GASright, we previously presented a computational and experimental structure-based analysis of 26 predicted dimers. We found that the contributions of van der Waals packing and Cei-H hydrogen bonding to stability, as inferred from the structural models, correlated well with relative dimerization propensities estimated experimentally with the in vivo assay TOXCAT. Here we test this model with a quan-titative thermodynamic analysis. We used Forster resonance energy transfer (FRET) to determine the free energy of dimeriza-tion of a representative subset of seven of the 26 original TOXCAT dimers using FRET. To overcome the technical issue arising from limited sampling of the dimerization isotherm, we introduced a globally fitting strategy across a set of constructs comprising a wide range of stabilities. This strategy yielded precise thermodynamic data that show strikingly good agreement between the original propensities and DG degrees of association in detergent, suggesting that TOXCAT is a thermodynamically driven process. From the correlation between TOXCAT and thermodynamic stability, the predicted free energy for all the 26 GASright dimers was calculated. These energies correlate with the in silico DE scores of dimerization that were computed on the basis of their predicted structure. These findings corroborate our original model with quantitative thermodynamic evidence, strengthening the hypothesis that van der Waals and Cei-H hydrogen bond interactions are the key modulators of GASright stability.