Thermodynamic analysis of the GAS right transmembrane motif supports energetic model of dimerization

Abstract 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 inter-helical 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 FRET to determine the free energy of dimerization of a representative subset of 7 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 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. Secondary Abstract We present a thermodynamic analysis of the dimerization of the GAS right motif, a common dimerization motif in membrane proteins. Previously, we found that the stability of GAS right is modulated by van der Waals packing and weak hydrogen bonds between Cα–H carbon donors and carbonyl acceptors on opposing helices. The experimental dimerization propensities were obtained with an in vivo assay. Here we assess this model quantitatively by measuring the free energy of dimerization of a subset of the original dimers. The thermodynamic data show strikingly good agreement between the original propensities and their ΔG° of association, confirming the model and strengthening the hypothesis that van der Waals and Cα–H hydrogen bond interactions are the key modulators of GAS right stability.