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

The GAS_right 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, GAS_right 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 Cα-H carbon donors and carbonyl acceptors on opposing helices (Cα-H···O=C). To understand the factors that modulate the stability of GAS_right, 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 Cα-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 quantitative thermodynamic analysis. We used Förster resonance energy transfer (FRET) to determine the free energy of dimerization 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 ΔG° 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 GAS_right dimers was calculated. These energies correlate with the in silico ΔE 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 Cα-H hydrogen bond interactions are the key modulators of GAS_right stability.