Complex interactions at the helix-helix interface stabilize the glycophorin A transmembrane dimer.

To explore the residue interactions in the glycophorin A dimerization motif, an alanine scan double mutant analysis at the helix-helix interface was carried out. These data reveal a combination of additive and coupled effects. The majority of the double mutants are found to be equally or slightly more stable than would be predicted by the sum of the energetic cost of the single-point mutants. The proximity of the mutated sites is not related to the presence of coupling between those sites. Previous studies reveal that a single face of the glycophorin A monomer contains a specific glycine-containing motif (GxxxG) that is thought to be a driving force for the association of transmembrane helices. Double mutant cycles suggest that the relationship of the GxxxG motif to the remainder of the helix-helix interface is complex. Sequences containing mutations that abolish the GxxxG motif retain an ability to dimerize, while a sequence containing a GxxxG motif appears unable to form dimers. The energetic effects of weakly coupled and additive double mutants can be explained by changes in van der Waals interactions at the dimer interface. These results emphasize the fact that the sequence context of the dimer interface modulates the strength of the glycophorin A GxxxG-mediated transmembrane dimerization reaction.

Sequence context modulates the stability of a GxxxG-mediated transmembrane helix-helix dimer.

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.

Thermodynamics of glycophorin A transmembrane helix dimerization in C14 betaine micelles.

We have used sedimentation equilibrium analytical ultracentrifugation to measure the free energy change for the glycophorin A transmembrane helix-helix dimerization in C14 betaine micelles. By varying the amount of micellar C14 betaine, we show that the protein association reaction in the micellar C14 phase behaves as an ideal-dilute solution. In this hydrophobic environment, the mole-fraction standard state free energy change for self-association of the SNGpA99 glycophorin A construct is -5.7 (+/-0.3, N=5) kcal mol(-1) at 25 degrees C. Compared with previous results carried out in C(8)E(5) micellar solutions, the free energy of dimerization is 1.3 kcal mol(-1) less favorable in C14 betaine micelles. In contrast, when considered on a per-interface basis, the formation of the glycophorin A transmembrane dimer in C14 betaine micelles may be more favorable than the association of several designed transmembrane peptides.