(545f) Perturbing the Hydrophobic Cores of Coiled-Coil Peptides with Non-Canonical Amino Acids | AIChE

(545f) Perturbing the Hydrophobic Cores of Coiled-Coil Peptides with Non-Canonical Amino Acids


Van Deventer, J. A. - Presenter, California Institute of Technology
Fisk, J. D. (. - Presenter, Colorado State University
Tirrell, D. A. - Presenter, California Institute of Technology and Joseph J. Jacobs Institute for Molecular Engineering for Medicine

Coiled-coil peptides are widely used model systems for investigating the stability and specificity of protein-protein interactions. Homodimeric interactions between molecules of A1, a model coiled-coil, in solution are mediated through the side chains of amino acids packed within the hydrophobic core of the coiled-coil and charged amino acid side chains flanking the core. Previous work has focused on studying the stability of A1 proteins after fluorinating residues within the hydrophobic core of the protein and has indicated that fluorinated coiled-coils possess greater stability than their hydrogenated counterparts.

However, studies aimed at addressing how fluorination affects protein stability have been limited by the fact that fluorination results in simultaneous changes to the hydrophobicity/fluorophilicity and sizes of amino acid side chains. Therefore, experimentally observed changes in stability could be the result of changes in either one of these side chain properties. To remedy this problem, we have been developing a hydrocarbon analog of leucine that possesses a side chain volume roughly equivalent to that of trifluoroleucine (Tfl). This volume equivalence can be achieved by substituting an ethyl group for one of the gamma methyl substituents of leucine, resulting in homoisoleucine (Hile). Hile can be prepared from commercial reagents in three steps. In medium lacking leucine, sub-millimolar concentrations of Hile support A1 protein synthesis in a leucine auxotrophic strain of E. coli that constitutively overexpresses leucyl-tRNA synthetase. Substitution at leucine positions by Hile in A1 has been investigated by mass spectrometry and is calculated to be greater than 95 percent. Studies of the biophysical properties of Hile-A1 are underway. Initial circular dichroism results indicate that the protein is highly helical at one degree Celsius. Furthermore, preliminary thermal denaturation studies suggest that the coiled-coil structure of Hile-A1 is more stable than the coiled-coil structure of leucine-A1. These results raise the possibility that the size increase that occurs upon fluorination is at least partially responsible for the increased stabilities observed in fluorinated coiled-coils.