Stabilization of Leucine Zippers with Trifluoroleucine A Molecular Dynamics Analysis

Yi Tang, Vaidehi Nagarajan, Jeremy Kua, Daniel T. Mainz, William A. Goddard III, David A. Tirrell

California Institute of Technology

Leucine zipper motifs had previously been engineered to facilitate the formation of reversible gelation networks. (Fig 1) The association strength and duration of the potential drug carriers depend strongly on the hydrophobic interactions between each zipper domains. We inserted trifluorolecuine, a close analog of leucine, in place of leucine in the zippers to achieve stronger hydrophobic interactions. (Fig 2, left panel) Experimental results suggest that indeed both the thermal and chemical stabilities were increased significantly upon such substitutions. (fig 2, right panel) We used molecular dynamics to understand the reasons for the increased association. Long time MPSIM simulation with solvation revealed that the increased hydrophobicity of the trifluoromethyl groups over the natural methyl groups significantly lowered the free energy of dimerization of the fluorinated coiled coils. The energy cost associated with the changes in electrostatic forces of the monomers, including those of disrupted hydrogen bonds upon folding, were also lowered compared to the wild type protein. The overall result is a 50% increase in the free energy of folding, agreeing to the phenomenon observed experimentally. The delicate balance between electrostatic forces and hydrophobic forces determines the stability of the coiled coils, as can be seen in the case of similar substitutions with hexafluoroleucines. While the hexamethyl groups contribute even more favorably to the hydrophobic driving force, the increased electrostatic energy cost of bring them from a random coil state to a folded state rendered the final structure less stable than that substituted with trifluroleucines. We wish to use these results to help us design gel helix domains with enhanced association strength.

Fig 1. The reversible hydrogel. Upper: The triblock copolymer in which the helix domains are of leucine zipper motifs. Lower: the helix domains can associate or dissociate reversibly under the change in temperature of pH.

Fig 2. Fluorinated leucine zippers. Left: Leucine zipper of study. The yellow residues are the leucines that were replaced by trifluoroleucine. They are hydrophobic redisdues that contribute to coiled coil stability. Right: The fluorianted dimers are much more stable compared to the wild type (CD spectroscopy)