This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Single transmembrane helices are rarely capable of performing biological functions. Instead, they form functional units after self-assembling into higher order structures. However, not all helices self-assemble. Consequently, it is necessary to understand sequence-specific interhelical recognition before we can predict the kinds of structures that can form in membranes. The simplest models for peptide association are helical dimers. Simple experimental models have recently been developed for helix associations in membranes. The best-studied system, both structurally and thermodynamically, is the 24-residue transmembrane region of glycophorin A (GpA). GpA forms non-covalent dimers through the reversible association of its membrane-spanning domain, which adopts an alpha-helical conformation. The structure has recently been determined by NMR spectroscopy of 40-residue peptides that contain the transmembrane segment, solvated in detergent micelles. This study also showed that the alpha-helices formed a right-handed, coil-coiled structure. We simulated the wild-type dimer of GpA in a lamella of dodecane, placed between two lamellae of water. The width of the dodecane layer was approximately the same as that of the hydrophobic core of a palmitoyloleylphosphatidylcholine (POPC) membrane. The starting structure was based on the NMR model of the dimer. The comparison between the calculated, time-averaged structure of the dimer after 30.0 ns of molecular dynamics trajectory and the nuclear Overhauser effect data of MacKenzie, et al., provided an assessment of the accuracy of our model and the potential energy functions utilized. The distance root mean square deviation was less than 1.5 Angstroms for the backbone atoms and 2.75 Angstroms for all atoms (Hydrogens excluded) and the monomers remained alpha-helical. Engelman, et. al, have observed interhelical close contacts between several Hydrogens bound to alpha Carbons and carbonyl Oxygens and argue that these can form weak hydrogen bonds that stablize the dimer. We observe that between 4 and 5 of these contacts are maintained over the course of the simulations and could contribute significantly to the stability of the dimer. The dissociation free energy of the GpA dimer in a detergent pentaoxyethylene (C8E5) has been estimated to be about 9.0 kcal/mol. It has been also demonstrated that single-residue mutations can markedly influence the free energy of association of the helices. Mutants in which either one of two leucine residues were substituted with alanine or glycine was substituted with isoleucine were found to be less stable than the wild-type dimer by 1-3 kcal/mol. All these residues are involved in interhelical interactions in the NMR determined model of GpA. We separated the helices in a series of molecular dynamics simulations using the distance separating the centers of mass of the two helices as the reaction coordinate. The free energy of dissociation was calculated using the Adaptive Biasing Force (ABF) method of Darve and Pohorille. The complete free energy pathway joining the dimer from 5.5 Angstroms at contact to dissociated monomers at 20 Angstroms was divided into a series of intermediate states corresponding to different values of the reaction coordinate. The free energy of dissociation is given as the sum of free energy differences between consecutive intermediate states. The computed free energy is approximately 10.0 kcal/mol. By comparison, the experimentally determined free energy of dissociation in the detergent pentaoxyethylene (C8E5) is equal to 9.0 kcal/mol. Since the molecular environments of GpA in the computational and experimental studies are different - ie. dodecane versus C8E5 - the free energies are not expected to be identical, but should be similar. Given that the volume accessible to the alpha-helical dimer is much smaller in C8E5 than in dodecane, which corresponds to a smaller entropic contribution, we expect that the free energy of dissociation should be higher in dodecane than in C8E5. Based on similar considerations, it is expected that the influence of the surroundings on the computed point mutations would, in principle, be limited due to compensation of entropic effects in the free energy differences. However, this may not be necessarily the case. The I76L point mutation was carried out computationally by decoupling the annihilation of the electrostatic and the van der Waals and internal parameter contributions. The free energy difference for the electrostatic term was 0 kcal/mol to within the statistical errors. The contribution due to the modification of the van der Waals parameters and the participating chemical bonds and valence angles, and, therefore, the total free energy difference, is equal to 0.4 kcal/mol, which is somewhat less than the experimental value of 1.7 kcal/mol. It should be emphasized, however, that, as the L-isoleucine is transformed into L-alanine, the disruptive effect of the mutation found experimentally in C8E5 should be less pronounced in dodecane, because of the greater volume accessible to the alpha-helices in that environment. The free energy change upon single-point mutation is fairly small. In contrast, the free energy of dissociation is large and positive. This might suggest that the dimer is strongly favored for both the wild-type and the mutants. This would indeed be the case if we only considered the equilibrium between the associated state and separated, transmembrane helices. However, this comparison is not appropriate because the transmembrane orientation of helices is not necessarily stable. The free energy of insertion of a helical peptide into the membrane is positive (unfavorable) and may be substantial. Thus, the equilibrium that needs to be considered is between the transmembrane dimer and the individual helices at the water-membrane interface. This equilibrium is governed by the balance between the unfavorable free energy of insertion into the membrane and favorable free energy of interhelical association. This balance could be subtle, and modest changes in either term could shift the equilibrium, possibly disrupting dimerization.
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