Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non‐fluorinated parallel dimeric coiled‐coils

Abstract

The α-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of coiled-coil motifs, including those modified by fluorination, several fluorinated and non-fluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiledcoil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER employed a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller Lornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine compared to 5,5,5,5′,5′,5′-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations * To whom correspondence should be addressed at the College of Pharmacy, University of Utah. Phone: (801) 587-9652, FAX: (801) 585-9119, e-mail: tec3@utah.edu. Supplementary MaterialSupplementary material is available that includes full sets of RMSd plots, results from force field comparisons, full description of new parameters, plots of water occupancy, more details regarding the free energy calculations, analysis of rotamer populations, and molecular graphics of the structural distortion in the ornithine substituted coiled-coil. suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5′,5′,5′-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may ...

Fluorinated amino acids: compatibility with native protein structures and effects on protein–protein interactions

“…Pendley et al recently presented a theoretical study of a highly fluorinated parallel coiled-coil heterodimer that contained 5 3 ,5 03 -F 6 Leu at all d-positions of both monomers. 45 Their simulations confirm the experimental findings that fluorination of Leu increases stability of the helical interactions thereby enhancing coiled-coil formation. Interestingly, their studies suggest that the dimer is not only stabilized by the increased hydrophobicity of the fluorinated side chain.…”

Effects of single substitutions with hexafluoroleucine and trifluorovaline on the hydrophobic core formation of a heterodimeric coiled coil

“…We found that a single substitution of Leu with 5 3 ,5' 3 -F 6 Leu leads to a slight increase in helicity and an increase in thermal stability, which can be explained by the highly favorable burial of the bulky hydrophobic fluorinated side chain. Thus, our experimental findings confirm the results of a theoretical study by Pendley et al, who found a free energy of hydration lower by 1.1 kcal mol -1 for 5 3 ,5' 3 -F 6 Leu compared to leucine [29]. The evaluation of high resolution X-ray structures of other peptides and proteins containing 5 3 ,5' 3 -F 6 Leu residues have shown that, although larger, these fluorinated residues preserve the shape of the A c c e p t e d M a n u s c r i p t 9 hydrophobic side chains that they substitute and thus are well accommodated within the coiled coil hydrophobic core with only minimal structural perturbations [7].…”

Synthesis of enantiomerically pure (2S,3S)-5,5,5-trifluoroisoleucine and (2R,3S)-5,5,5-trifluoro-allo-isoleucine

“…F 6 Leu is similar to Aha in hydrophobicity, while exhibiting a much larger volume. In a free energy perturbation study, the hydration energy of F 6 Leu was shown to be 1.1 kcal/mol higher than that of leucine [29]. This, together with our previous and new findings, suggests that there are two factors determining the overall hydrophobicity of fluorinated amino acids [26].…”